pedro beltran-alvarez - uni-hannover.de · during my ph.d, and all these people have contributed to...

Kinetic studies on the actinorhodin

polyketide synthase

Pedro Beltran-Alvarez

A dissertation submitted to the University of Bristol

in accordance with the requirements of the degree of

Doctor of Philosophy

in the School of Chemistry, Faculty of Science.

April 2008

Word count: 41,919

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ABSTRACT

The actinorhodin (act) minimal polyketide synthase (PKS) from Streptomyces coelicolor

consists of three proteins: an acyl carrier protein (ACP), and two β-ketoacyl ACP synthase

components known as KSα and KSβ. The act minimal PKS catalyzes at least 18 separate

reactions, which can be divided into loading, initiation, extension, and cyclization and

release phases.

Quantitative kinetic assays were developed and used to measure individual catalytic

(kcat) and Michaelis (KM) constants for loading, initiation and extension steps. In vitro, the

reaction between malonyl CoA and ACP to form malonyl ACP (loading) is the rate-

limiting step (kcat = 0.49 min-1, KM = 207 µM). This reaction increases 5-fold in rate in the

presence of KSα/KSβ (kcat = 2.3 min-1) with unchanged KM. In the presence of S. coelicolor

malonyl CoA: ACP transacylase (MCAT), formation of malonyl-ACP is even faster: under

these conditions, it appears that decarboxylation of malonyl-ACP to form acetyl-ACP

(initiation) is the rate-limiting step (kcat = 20.6 min-1, KM = 2.4 µM). When an excess of

acetyl ACP is supplied, chain extension becomes rate limiting (kcat ~ 60 min-1). No ACP-

bound intermediates could be observed, suggesting that fully extended chains do not

accumulate, so cyclization and release are likely still faster than chain extension.

Protein-protein interactions among the components of the act minimal PKS were

investigated. It appears that KSα and KSβ have two different binding sites for ACP,

because inactive apo-ACP acts as a mixed inhibitor of malonyl-ACP binding to KSα/KSβ.

E47 and E53 in ACP are important residues for ACP: KSβ interactions. At least E47

appears to be important for ACP: KSα binding as well.

The quaternary structure of the active KSα/KSβ is most likely tetrameric.

Decarboxylation of malonyl-ACP by KSβ may be accompanied by a conformational

change in the structure of the tetramer that renders KSα ready for extension.

Addition of act KR to the minimal PKS does not affect the rates of loading, initiation or

extension, suggesting that ketoreduction is faster than chain extension. KR does not seem to

interact with KSα/KSβ, and probably acts before the first cyclisation of the polyketide

chain.

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To Carlos, Laura, Pedro, Pilar and Sonsoles

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ACKNOWLEDGEMENTS

Four years ago, Russell Cox invited me to join his group and do a Ph.D in organic and

biological chemistry, a field that is, at least at first sight, strange for a chemical engineer.

Having today arrived at the time of writing these acknowledgements, it is Russell the first

person I want to mention, because without his initial support, this moment would have

never come. Thanks Russell for the confidence you had on me from day one, for your

friendship, and for your constant support and supervision during my PhD.

My arrival at Bristol was also made possible by my family and my friends in Santander,

who understood my decision of leaving home and have made distances shorten by

travelling to and fro on every big occasion. Thanks guys for making me feel as I had never

gone whenever I was back home.

I spent three years in Bristol (2004-2007). I was welcomed by a group of people completely

different than the one I left: Kirstin, Ana, Dougal, Ludo and Jenny left soon; Pakorn, Deaw,

Chris and Chris, Kate, Elaine, Joel, Simon, David and particularly Bei, who took care of me

in the lab during my first months, and the very special Deirdre, Ursula and Song, made

Bristol a great place to live during the first years. Huge thanks also to the people that started

at the same time as me, that is you, Laura, Ellen, Tom, Marc, Carlo, Alexa, Eliza, and

specially my girlfriend Laura, because it is you with whom I shared most of the time during

these three years. You all, together with James (Dr. Goldfish), Mateusz (Prof. Scorpio), Liz

(Dr. Pepper), Andy, Ahmed, Rozida, Zafar, Persefoni and Anna deserve my gratitude for

having made these three years the best of my life. I will never forgive you.

I would also like to thank John Crosby for useful discussions, Paul Gates for help with

ESMS, and Chris, Pakorn, Eliza, Chris, Marc, Joel, Simon, Bei, Song, Laura, as well as

Gus Cameron, Tony Clarke and Annela Seddon in Biochemistry, for either materials,

training or help with sample analysis (mass spec, haddock, stopped-flow, fplc, hplc, plate

reader, pcr…).

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Also, I am grateful to my examiners Matt Crump and Peter Leadlay for reading my thesis

and housing my viva, and to all the BRISENZ staff, i.e. Russell Cox, John Crosby, Matt

Crump, Andrea Hadfield, Adrian Mulholland, Tom Simpson and Chris Willis for

organizing the Marie Curie Programme of the European Commission, which is

acknowledged for funding (Marie Curie EST Centre BRISENZ, Contract No.: MEST-CT-

2004-504051).

Last, I thank you, the reader, for taking the time to read these acknowledgements, which are

full of names you may or may not recognize -those were the names I pronounced most

during my Ph.D, and all these people have contributed to what follows. Hope you will find

the read worthwhile.

Heidelberg, August 16th, 2008.

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AUTHOR’S DECLARATION

I declare that the work in this dissertation was carried out in accordance with the

Regulations of the University of Bristol. The work is original except where indicated by

special reference in the text and no part of the dissertation has been submitted for any other

academic award. Any views expressed in the dissertation are those of the author and in no

way represent those of the University of Bristol. The dissertation has not been presented to

any other University for examination either in the United Kingdom or overseas.

Pedro Beltran-Alvarez, August 16th, 2008.

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TABLE OF CONTENTS

1. Introduction ................................................................................................................1

1.1. Natural products ......................................................................................................................1

1.2. Polyketide biosynthesis............................................................................................................1 1.2.1. Polyketide biosynthesis is similar to fatty acid biosynthesis .......................................................... 2 1.2.2. Type II polyketide synthases ............................................................................................................ 5

1.2.2.1. The actinorhodin polyketide synthase...................................................................................... 6 1.2.3. The actinorhodin minimal PKS ........................................................................................................ 9

1.2.3.1. The actinorhodin Acyl Carrier Protein (ACP).......................................................................10 1.2.3.2. S. coelicolor malonyl-CoA: ACP transacylase (MCAT)......................................................12 1.2.3.3. The actinorhodin ketosynthase complex (KSα/KSβ). ..........................................................14 1.2.3.4. The Stanford model for the act minimal polyketide synthase in vitro .................................17 1.2.3.5. The Bristol model for the actinorhodin minimal polyketide synthase in vitro ....................19

1.2.4. Type III polyketide synthases .........................................................................................................21 1.2.5. Type I modular polyketide synthases.............................................................................................22

1.2.5.1. The 6-deoxyerythronolide B (6-dEB) polyketide synthase. .................................................23 1.2.6. Type I iterative polyketide synthases .............................................................................................24

1.3. Structure of the mammalian fatty acid synthase as a model for polyketide synthases.

26 1.3.1.1. Initial experiments: the linear model (1970s-1990s).............................................................26 1.3.1.2. Building of a 2D model (1990s) .............................................................................................26 1.3.1.3. 3D models for FAS and PKS megasynthases (1990s-2000s)...............................................28

1.4. Fundamentals of enzyme kinetics ........................................................................................30 1.4.1. Rate equations in enzyme kinetics..................................................................................................30 1.4.2. Kinetics of the act minimal PKS ....................................................................................................33

1.5. Aim of the project...................................................................................................................35

2. Studies on Acyl Carrier Proteins...............................................................................36

2.1. Protein-protein interactions in Type II FAS and PKS.....................................................37

2.2. Purification of acyl carrier proteins ....................................................................................40

2.3. Reduction of ACP dimers .....................................................................................................40

2.4. Studies on the self-malonylation activity of act holo-ACP. .............................................42

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2.4.1. Development of the α-ketoglutarate dehydrogenase assay to measure the rate of self-

malonylation ...................................................................................................................................................43 2.4.1.1. Development of the method to study the self-malonylation of act holo-ACP ....................45 2.4.1.2. Kinetics of the self-malonylation of act holo-ACP ...............................................................45

2.4.2. Actinorhodin minimal PKS to measure self-malonylation rate....................................................47 2.4.2.1. Development of the method....................................................................................................47 2.4.2.2. Kinetics of the self-malonylation of act holo-ACP ...............................................................50

2.5. Studies on the mechanism of self-malonylation of ACP ..................................................51 2.5.1. Mechanism of acceleration of self-malonylation rate by KSα/KSβ. ...........................................53

2.6. Discussion ................................................................................................................................56 2.6.1. Studies on the quality of ACP prior to reaction.............................................................................56 2.6.2. Both malonate and CoA are important for binding onto ACP .....................................................57 2.6.3. KSα/KSβ accelerates malonylation of ACP..................................................................................59

2.6.3.1. KSα/KSβ as a potential acyl transferase................................................................................60 2.6.3.2. KSα/KSβ activates holo-ACP towards self-malonylation ...................................................61 2.6.3.3. Insights into ACP-KSα/KSβ interactions leading to enhanced self-malonylation activity.

65

3. Initiation of polyketide synthesis. ..............................................................................67

3.1. Kinetics of KSβ can be studied in the presence of MCAT ..............................................74 3.1.1. MCAT accelerates the rate of malonylation of holo-ACP............................................................74 3.1.2. Initiation of polyketide synthesis is slower than chain elongation...............................................77

3.2. Interaction of ACP and KSβ ................................................................................................78

3.3. Discussion ................................................................................................................................79

4. Chain elongation and cyclisation and release from the PKS ....................................82 4.1.1. Kinetics of FAS systems .................................................................................................................83 4.1.2. Kinetics of Type III PKS.................................................................................................................84 4.1.3. Kinetics of Type I modular PKS (DEBS) ......................................................................................86

4.2. Kinetics of the rate-limiting reaction catalyzed by KSα ..................................................87 4.2.1. Attempts to detect polyketide intermediates..................................................................................88 4.2.2. Kinetics of the elongation step catalyzed by KSα.........................................................................89

4.3. Discussion ................................................................................................................................91

5. Stoichiometric analysis of the act minimal PKS .......................................................95

5.1. Stoichiometry of the KSα/KSβ complex –assays with mutant KSα/KSβ ....................96

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5.1.1. Kinetic study of a mutant CA KSα/KSβ complex ........................................................................99

5.2. Stoichiometry of the ACP: KSα/KSβ complex – inhibition of KSα/KSβ by apo-ACP.

101

5.3. Discussion ..............................................................................................................................104 5.3.1. Stoichiometry of ACP: KSα/KSβ complexes .............................................................................107

6. Kinetics of an extended act minimal PKS ...............................................................110

6.1. Structure and chemical mechanism of KR ......................................................................117

6.2. Kinetics of extended Type II minimal PKS......................................................................118

6.3. Kinetic analysis of production of mutactin by an extended act minimal PKS ...........119 6.3.1. Self-malonylation of ACP in the presence of KR .......................................................................123 6.3.2. Addition of MCAT to extended minimal PKS assays ................................................................125 6.3.3. Addition of acetyl-ACP to MCAT-supplemented, extended minimal PKS assays ..................126

6.4. Discussion ..............................................................................................................................126

7. Conclusions.............................................................................................................129

7.1. Self-malonylation of holo-ACP is aided by KSα/KSβ ....................................................130

7.2. Quaternary structure of KSα/KSβ and ACP: KSα/KSβ complexes ..........................133

7.3. Model for an extended act minimal PKS..........................................................................135

8. Experimental procedures ........................................................................................138

8.1. Bacterial strains and plasmids ...........................................................................................138 8.1.1. Escherichia coli..............................................................................................................................138 8.1.2. Streptomyces coelicolor ................................................................................................................138

8.2. DNA techniques ....................................................................................................................139 8.2.1. Site directed mutagenesis ..............................................................................................................139 8.2.2. Plasmid DNA isolation and characterization ...............................................................................140

8.3. Bacterial culture tecniques .................................................................................................140 8.3.1. Liquid media ..................................................................................................................................140

8.3.1.1. SOC medium..........................................................................................................................140 8.3.1.2. Luria-Bertani (LB) medium..................................................................................................141 8.3.1.3. Super-YEME (SY) medium..................................................................................................141

8.3.2. Solid media ....................................................................................................................................141 8.3.2.1. Luria-Bertani agar (LB agar) growth medium.....................................................................142 8.3.2.2. Mannitol Soya Flour Medium (SFM) ..................................................................................142

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8.3.2.3. R5 Medium ............................................................................................................................142

8.4. Extraction and purification of SEK4 and SEK4b...........................................................143 8.4.1. Growth of bacteria and extraction of polyketides .......................................................................143 8.4.2. Liquid Chromatography/ Mass Spectrophotometry (LC/MS) analysis .....................................143 8.4.3. Preparative HPLC for purification of SEK4 and SEK4b............................................................144

8.5. Growth of bacteria and purification of proteins .............................................................144 8.5.1. General methods and equipment ..................................................................................................144

8.5.1.1. Centrifugation ........................................................................................................................144 8.5.1.2. Shakers ...................................................................................................................................144 8.5.1.3. UV Spectrophotometer..........................................................................................................144 8.5.1.4. Plate reader.............................................................................................................................145 8.5.1.5. Sonication...............................................................................................................................145 Cell-free extracts were obtained after lysing the cells by sonication (MSE Soniprep 150). Sonication

of E.coli was executed in 5 short bursts of 30 seconds with 1.5 min ice-cooling in between. S.

coelicolor was sonicated 10 times for 30 seconds. ...............................................................................145 8.5.1.6. Buffers ....................................................................................................................................145 8.5.1.7. NTA-His-Bind nickel affinity chromatography ..................................................................145 8.5.1.8. Fast protein liquid chromatography (FPLC)........................................................................146 8.5.1.9. Freeze-Drying ........................................................................................................................146 8.5.1.10. Sodium dodecyl sulphate Polyacrylamide gel electrophoresis (SDS-PAGE).................146

8.5.2. Growth of E. coli, and expression and purification of ACP, ACPS and MCAT ......................147 8.5.2.1. Purification of ACP ...............................................................................................................148 8.5.2.2. Purification of S. coelicolor his6-ACPS and act his6-KR....................................................148 8.5.2.3. Purification of S. coelicolor his6-MCAT .............................................................................149

8.5.3. Growth of S. coelicolor, expression and purification of his6-act-KSα/KSβ. ............................149 8.5.3.1. Growth of bacteria and expression of KSα/KSβ.................................................................149 8.5.3.2. Purification of KSα/KSβ ......................................................................................................149

8.6. Protein characterization methods......................................................................................150 8.6.1. Protein Quantification ...................................................................................................................150

8.6.1.1. Bradford Assay ......................................................................................................................150 8.6.1.2. Bicinchonic Acid Assay (BCA) ...........................................................................................150 8.6.1.3. Extinction coefficient of proteins. ........................................................................................151 8.6.1.4. Electro-Spray Mass Spectrometry (ESMS) .........................................................................151

8.7. Protein assays........................................................................................................................151 8.7.1. Phosphopantetheninylation of E47V apo-ACP to produce E47V holo-ACP............................152 8.7.2. Reduction of ACP dimers. ............................................................................................................152 8.7.3. Acylation of holo-ACP..................................................................................................................152

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8.7.4. Study of the self-malonylation of ACP using α-ketoglutarate dehydrogenase (KGDH) .........152 8.7.5. Minimal actinorhodin polyketide synthase assays and HPLC analysis of SEK4 and SEK4b .153 8.7.6. Kinetic studies on the actinorhodin minimal polyketide synthase .............................................154

8.7.6.1. Calibration of the method .....................................................................................................154 8.7.6.2. Minimal polyketide synthase assays to measure self-malonylation of holo-ACP ............154 8.7.6.3. Minimal polyketide synthase assays to measure inhibition of self-malonylation of holo-

ACP by CoA-SH .....................................................................................................................................155 8.7.6.4. Minimal polyketide synthase assays to measure the kinetics of KSβ ................................155 8.7.6.5. Minimal polyketide synthase assays to measure the kinetics of KSα ...............................155 8.7.6.6. Minimal polyketide synthase assays to measure inhibition of KSα/KSβ by apo-ACP....155 8.7.6.7. Minimal polyketide synthase assays to measure the kinetics of CA KSα/KSβ ................156 8.7.6.8. Data analysis ..........................................................................................................................156

References………………………………...…………………………………………..157

Appendices……………………………………………………...…………………….178

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LIST OF FIGURES Figure 1.1 Polyketides exhibit wide structural variety and pharmacological activities....................................... 2 Figure 1.2 Malonyl CoA is an important acyl carrier molecule. ........................................................................... 5 Figure 1.3 Natural products produced by Streptomyces species, derived from Type II PKS. The starter unit is

shown in red ............................................................................................................................................................... 6 Figure 1.4 The core of the actinorhodin (act) PKS genes.22 ................................................................................... 7 Figure 1.5 Structure of actinorhodin apo-ACP (PDB file: 2AF8), coloured from N-terminus (blue) to C-

terminus (red). ..........................................................................................................................................................11 Figure 1.6 Crystal structure of S. coelicolor MCAT, highlighting the two subdomains (blue and red) and the

catalytic active site S97 (green). PDB code: 1NM2. .............................................................................................13 Figure 1.7 Structure of KSα, showing from top to bottom the typical five layered α-β-α-β-α bundle fold of

thiolase enzymes. The active site cysteine is highlighted. PDB file: 1TQY..........................................................16 Figure 1.8 Primary sequence alignment of the active site (green star) region of a range of FAS ketosynthases

as well as KSα and KSβ from various PKS. For a complete sequence alignment see Appendix II. ..................17 Figure 1.9 Gen sequence of a DEBS PKS module. KS = ketosynthase, AT = acyl transferase, DH =

dehydratase, ER = enoyl reductase, KR = ketoreductase, ACP = acyl carrier protein. DH and ER are only

present in module 4. .................................................................................................................................................24 Figure 1.10 Head-to-tail model and linear arrangement of a dimeric FAS. Single headed arrows indicate the

order of domains in the primary structure (N-terminus to C-terminus). Blue, double headed arrows indicate

the interaction between ACP and KS domains. .....................................................................................................26 Figure 1.11 Complementation studies with mutant FAS. The inactivated domain is shown by X. A. Active

condensation intersubunit; B. Active condensation intrasubunit; C. Active acyl transfer intersubunit; D.

Active acyl transfer intrasubunit; E. No dehydration intersubunit; F. Active dehydration intrasubunit.83.......27 Figure 1.12 2-D model for the topology of Type I mammalian FAS. Single headed arrows indicate the order

of domains in primary sequence from N-terminus to C-terminus. Blue, double headed arrows indicate

interaction between ACP and KS, and between ACP and MAT.86 .......................................................................28 Figure 1.13 Structure of Type I FAS megasynthase (PDB file: 2CF2). Yellow, AT; red & green, the two

monomers of KS; blue, DH; magenta, homodimeric ER; orange, KR. The proposed reaction chambers are

marked with a star. ..................................................................................................................................................29 Figure 2.1 Primary sequence alignment of FAS and PKS ACP (green star, the PP attachment serine). .........39 Figure 2.2 Mass spectra of A. act ACP (expected, 9441 Da); B. dps ACP (expected 9603 Da); C. gris ACP

(expected, 9884 (-Me) and 10016 (+Me) Da); D. E47A-ACP (expected, 9383 Da); E. E47V-ACP (expected,

9412 Da); F. E53A-ACP (expected, 9383 Da) and G. R72A-ACP (expected, 9357 Da)....................................40 Figure 2.3 Mass spectrum of the reference act holo-ACP dimer after incubation with TCEP. Expected masses,

9441 Da (monomer), 18882 Da (dimer).................................................................................................................41 Figure 2.4 A. Incubation of malonyl-ACP with DTT. B. Incubation of malonyl-ACP with TCEP. Expected

masses: 9441 Da (holo-ACP) and 9527 Da (malonyl-ACP). ...............................................................................42 Figure 2.5. Raw data for the KGDH assay at initial holo-ACP concentrations of 15 µM (squares), 30 µM

(triangles) and 50 µM (circles). Malonyl CoA concentration was fixed at 1mM................................................46

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Figure 2.6 Determination of Michaelis-Menten kinetic parameters of the self-malonylation of holo-ACP by

the KGDH coupling system. A. Plot of reaction rate vs. substrate concentration and hyperbolic fit. B. Hanes

linear plot of the data. .............................................................................................................................................47 Figure 2.7. Increase in the absorbance at 293 nm in act minimal PKS assays. Holo-ACP and malonyl CoA

concentrations were fixed at 80 and 1000 µM respectively. KSα/KSβ was 0.3 µM (diamonds), 0.5 µM

(triangles) and 1 µM (circles). ................................................................................................................................49 Figure 2.8 Dependence of initial rates of octaketide production on the concentration of KSα/KSβ at varying

ACP concentrations: squares, 10 µM; crosses, 25 µM; triangles, 50 µM; circles, 80 µM ACP.......................50 Figure 2.9 Determination of Michaelis-Menten kinetic parameters for the self-malonylation of holo-ACP by

the KSα/KSβ coupling system. A. Plot of reaction rate vs. substrate concentration and hyperbolic fit. B. Hanes

linear plot of the data. .............................................................................................................................................51 Figure 2.10 Interaction between arginine residues and malonate units.57 ..........................................................52 Figure 2.11 Determination of Kic and Kiu for the inhibition self-malonylation of ACP by coenzyme A (open

triangles, 800 µM; filled triangles, 400 µM; open circles, 200 µM; filled circles, 100 µM). ............................53 Figure 2.13 Self-malonylation of the reference act ACP (circles) and E47A ACP (open circles) as measured

by ESMS....................................................................................................................................................................54 Figure 2.14 HPLC analysis of SEK4 and SEK4b produced in 2 h by KSα/KSβ (0.3 µM) and a series of ACP

(50 µM). From bottom to top, the reference act ACP, dps ACP, E53A ACP, R72A ACP, E47A ACP, E47V

ACP, gris ACP. Yields after 2 h were 5.5, 2.3, 2.0, 2.3, 0.6, 0.1 and 0.04 pmol SEK4 and SEK4b, respectively.

...................................................................................................................................................................................55 Figure 2.15 A. Affected residues upon binding of malonyl CoA onto ACP (yellow). B. All affected residues are

solvent-exposed (blue).134 ........................................................................................................................................58 Figure 2.16 Ribbon representation of the major (A. PDB file 2FQ0) and minor (B. PDB file 2FQ2)

conformers of P. falciparum FAS ACP. Residues D57 to A60 are highlighted in magenta. ..............................61 Figure 2.17 ACP and PCP activate their substrates (for instance, malonate and alanine) as thiolesters........63 Figure 2.18 Ribbon structures of TyrC3-PCP in its A state (A. PDB file, 2GDY), A/H state (B. PDB file

2GDW) and H state (C. PDB file 2GDX). Helices are numbered in each structure...........................................64 Figure 3.1 Mass spectrum of MCAT (expected, 34113 Da); also detected as the potassium adduct (at 34152

KDa)..........................................................................................................................................................................74 Figure 3.2 A. Comparison of act PKS ‘minimal’ assays (open circles) and MCAT-supplemented assays (filled

circles). B. Hanes linear plot of the MCAT-supplemented rate data. ..................................................................76 Figure 3.3 Upon addition of MCAT, overall reaction rates become linearly dependent on KSα/KSβ

concentrations. .........................................................................................................................................................76 Figure 3.4 ESMS of acetyl-ACP (expected 9483 Da), also detected as the potassium adduct (expected 9522

Da). ...........................................................................................................................................................................77 Figure 3.5 Change in reaction rate upon titration of acetyl-ACP in MCAT-supplemented act minimal PKS.

KSα/KSβ and malonyl-ACP concentration were 0.25 µM and 20 µM respectively. ..........................................78 Figure 4.1 ESMS analysis of the ACP species in minimal PKS assays done in the presence of MCAT and

external acetyl-ACP. Expected masses: 9527 Da (malonyl-ACP), 9483 Da (acetyl-ACP)................................89

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Figure 4.2. Evolution of the production of polyketides with time at saturating acetyl-ACP concentration and

2.5 µM malonyl-ACP (triangles), 5 µM malonyl-ACP (circles) and 10 µM malonyl-ACP (crosses). ..............90 Figure 5.1 Rate of polyketide production by the act PKS (supplemented with MCAT) using CQ KSα/KSβ

(0.25 µM) at a range of AQ KSα/KSβ concentrations. .........................................................................................98 Figure 5.2 Kinetics of the decarboxylation of malonyl-ACP by CA KSα/KSβ. A. Plot of reaction rate vs.

substrate concentration and hyperbolic fit. B. Hanes linear plot of the rate data. .............................................99 Figure 5.3 Rate of polyketide production by the act PKS (supplemented with MCAT) using CA KSα/KSβ (1

µM) at a range of AQ KSα/KSβ concentrations. .................................................................................................100 Figure 5.4 Mass spectrum of act C17S apo-ACP (expected, 9101 Da, also observed as the sodium and

potassium adduct at 9124 and 9140 Da, respectively)........................................................................................103 Figure 5.5 Inhibition of KSα/KSβ by apo-ACP at holo-ACP concentrations of 2.5 µM (full squares), 5 µM

(circles), 7.5 µM (squares), 15 µM (full circles) and 25 µM (triangles). ..........................................................103 Figure 5.6 Re-plot of data in Figures 5.1 and 5.3.Titration of AQ in assays performed with WT KSα/KSβ

(blue, 0.25 µM concentration) and CA (red, 1 µM concentration). ...................................................................107 Figure 6.1 Structure of a monomer of act KR, showing the β-sheet core (blue) and the catalytic residues

N114, S144, Y157 and K161 (yellow). PDB file: 1W4Z......................................................................................117 Figure 6.2 Mass Spectrum of his6-KR (expected: 29291 Da, observed: 29299 Da) and α-N-6-

phosphogluconoylation of the His-tag (expected: 29467 Da, observed: 29477 Da). .......................................120 Figure 6.3 LC/MS analysis of mutactin. A. Chromatogram showing the mutactin peak at 26.40 min. B.

Individual mass chromatogram at 303 Da (expected mass of [M+H]+). ..........................................................121 Figure 6.4 HPLC chromatograms at 280 nm showing the biosynthesis of SEK4 19, SEK4b 27 and mutactin 20

at fixed concentrations of malonyl CoA (1 mM), holo-ACP (50 µM) and KSα/KSβ (0.6 µM) and a range of

KR concentrations..................................................................................................................................................121 Figure 6.5 Effect of increasing concentrations of KR on the ratio mutactin: (SEK4 + SEK4b). .....................122 Figure 6.6. HPLC chromatograms at 280 nm of the time course study of polyketide production (SEK4 19,

SEK4b 27 and mutactin 20) in the absence (A) and presence (B) of KR (10 µM). ...........................................123 Figure 6.7 HPLC chromatograms at 280 nm showing polyketide production (SEK4 19, SEK4b 27 and

mutactin 20) upon titration of KSα/KSβ in extended minimal PKS assays at fixed KR concentration (10 µM).

.................................................................................................................................................................................124 Figure 6.8 Determination of kinetic parameters for the self-malonylation of holo-ACP in the presence of KR

by the KSα/KSβ coupling system. .........................................................................................................................125 Figure 6.9 Determination of kinetic parameters in MCAT-supplemented minimal PKS assays in the presence

of KR. ......................................................................................................................................................................126 Figure 7.1 Comparison of the behavior of MCAT-catalyzed malonylation of holo-ACP vs self-acylation of act

PKS holo-ACP. Linear plots show how the rate of self-malonylation of holo-ACP in the presence of KSα/KSβ

varies with ACP concentration at two concentrations of malonyl CoA (rate data from Figure 2.6). Curves

show the effect of saturation catalysis by MCAT at differing MCAT concentrations (for simulated MCAT with

KM of 60 µM for ACP and kcat of 450 s-1 at saturating malonyl CoA). Grey horizontal line shows demand for

malonyl ACP by KSα/KSβ at 0.75 µM. ................................................................................................................132

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Figure 7.2 Model for ACP: KSα/KSβ that contemplates four molecules of ACP per tetramer KSα/KSβ. .....134

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LIST OF SCHEMES Scheme 1.1 Archetypical two-steps mechanism for the generation of a new carbon-carbon bond by FAS and

PKS: 1. Decarboxylation of activated carboxylic acid; 2. Claisen condensation. ................................................ 3 Scheme 1.2 Processing of the β-carbon after Claisen condensation in FAS. ........................................................ 4 Scheme 1.3 Proposed early steps in the biosynthesis of actinorhodin ................................................................... 7 Scheme 1.4 Proposed later steps in actinorhodin biosynthesis.29........................................................................... 9 Scheme 1.5 Biosynthesis of SEK4 and SEK4b by S. coelicolor CH999/pSEK4, which encodes the act minimal

PKS. ..........................................................................................................................................................................10 Scheme 1.6 Act holo-ACP consists of apo-ACP plus a PP cofactor (shown in red) transferred by ACPS from

CoA to Ser42 in ACP.................................................................................................................................................12 Scheme 1.7 MCAT catalyzes the malonylation of ACP in FAS systems...............................................................12 Scheme 1.8 Two-step mechanism for MCAT-catalyzed transfer of malonate to FAS-ACP. A. Loading of

malonate onto MCAT; B. Transfer of malonate to holo-ACP and regeneration of MCAT. ...............................13 Scheme 1.9 Reaction catalyzed by a general thiolase from a thiolester substrate.50 ..........................................14 Scheme 1.10 Mechanism of two-step decarboxylative Claisen condensation in thiolases (act KSα numbering),

proposed.51................................................................................................................................................................15 Scheme 1.11 The Stanford model for production of SEK4 and SEK4b by the act minimal PKS.32 A. Loading of

ACP is catalyzed by MCAT. B. Decarboxylation of malonyl-ACP is catalyzed by KSα, which subsequently

takes the acetyl starter unit. C. Claisen condensation and elongation of the polyketide chain. D. The

polyketide chain extrudes in a tunnel formed in the interface between KSα and KSβ, and finally cyclizes and it

is released from the PKS. ........................................................................................................................................18 Scheme 1.12 Self-malonylation of holo-ACP .........................................................................................................19 Scheme 1.13 Model for Type II PLS. Progressing clockwise, self-malonylation and malonyl transfer provide

the malonate substrate for initiation of polyketide synthesis by KSβ and chain elongation by KSα .................20 Scheme 1.14 Biosynthesis of chalcone and resveratrol (a stilbene) by chalcone (CHS) and stilbene (STS)

synthases.67 ...............................................................................................................................................................22 Scheme 1.15 Biosynthesis of 6-dEB by the Type I modular DEBS. Domains within each module follow the

order KS-AT-(DH)-(ER)-KR-ACP ..........................................................................................................................24 Scheme 1.16 Mechanism for enzyme-catalyzed reaction proposed by Michaelis and Menten. S = substrate, E

= enzyme, P = product. ...........................................................................................................................................31 Scheme 1.17 Briggs-Haldane mechanism. The concentration of the different species is shown underneath. ..31 Scheme 1.18 Biosynthesis of octaketides by the act minimal PKS. A. Loading of holo-ACP with malonate. B.

Initiation of polyketide synthesis by formation of acetyl-ACP. C. Elongation of the polyketide chain. D.

Formation of SEK4 and SEK4b in vitro. E. Further tailoring reactions lead to actinorhodin in vivo.1............34 Scheme 2.1 Disulphide formation between the thiols of holo-ACP......................................................................36 Scheme 2.2 Reaction scheme of KAS III. A. Condensation of acetyl CoA and malonyl-ACP to form

acetoacetyl-ACP. B. The same reaction is possible using malonyl CoA as a substrate instead of malonyl-ACP.

...................................................................................................................................................................................38 Scheme 2.3 Reaction scheme of the KGDH assay. ................................................................................................44

xvii

Scheme 2.4 Quantitative KGDH assay for CoA-SH. NADH production is followed spectrophotometrically. .45 Scheme 2.6 Two-step mechanism for the actinorhodin minimal PKS. 1. Self-malonylation of holo-ACP. 2.

Polyketide synthesis from malonyl-ACP.................................................................................................................50 Scheme 2.8 DTT reduces malonyl-ACP concentration by transthiolesterification .............................................57 Scheme 2.9 Mechanism that produces mixed inhibition of self-malonylation by CoA-SH. ................................58 Scheme 3.1 Initiation of fatty acid synthesis in E. coli, catalyzed by KASIII.144..................................................67 Scheme 3.2 Proposed mechanism for starter unit specificity in dps PKS. A. Loading of DpsC with propionate.

B. Initiation of polyketide synthesis. C. Elongation of the polyketide chain.149...................................................69 Scheme 3.3 Priming of the enc PKS with the benzoate starter unit.126.................................................................70 Scheme 3.4 Two possible models for transfer of acetyl-ACP from the KSβ to KSα active sites. .......................72 Scheme 4.1 Reactions catalyzed by KSα: A. Priming; B. Chain extension. C. Acyl transfer between the PP

thiol of ACP and the cysteine active site in KSα....................................................................................................83 Scheme 4.2 Reaction catalyzed by the pentaketide resorcylic synthase from Neurospora crassa .....................84 Scheme 4.3 Reactions catalyzed by A. Module 3 + TE; B. Modules 2, 5 and 6 + TE. .......................................86 Scheme 5.1 Dimer-tetramer KSα/KSβ equilibrium. ..............................................................................................96 Scheme 5.2 Hypothetical use of AQ KSα/KSβ to increase the rate of KSβ-mediated decarboxylation of

malonyl-ACP. KSα (red) and KSβ (green) are depicted by their corresponding active sites (see text). ...........97 Scheme 5.3 General mixed inhibition mechanism for apo-ACP as an inhibitor of KSα/KSβ ..........................102 Scheme 5.4. The addition of AQ affects the composition of A. CQ KSα/KSβ tetramers; B. CA KSα/KSβ

tetramers. ................................................................................................................................................................105 Scheme 6.1 Complementation of S. galilaeus with act KR resulted in production of a reduced polyketide (the

propionate starter unit is shown in red).198 ..........................................................................................................110 Scheme 6.2 Effect of addition of KR to act minimal PKS in in vivo experiments. .............................................111 Scheme 6.3 Production of 72 and 71 by S. galilaeus HO61 and S. galilaeus HO61 complemented with KR,

respectively. The propionate starter unit is shown in red. ..................................................................................112 Scheme 6.4 Model proposed by the group of Tsai, in which the substrate for act KR is the bicyclic

intermediate 82.206 .................................................................................................................................................114 Scheme 6.5 Proposed biosynthesis of BSM1 and BSM3.207 ................................................................................115 Scheme 6.6 Biosynthesis of wailupemycin D-G by the enc minimal PKS. Wailupemycin D: R1 = OH, R2 = Ph;

Wailupemycin E: R1 = Ph, R2 = OH.203,208 ...........................................................................................................116 Scheme 6.7 Proton relay mechanism in the act KR active site (blue arrows) showing NADPH (green), the

polyketide substrate (red) and the active site residues (black).205......................................................................118 Scheme 6.8 Biosynthesis of decaketides by tcm PKS...........................................................................................119

xviii

LIST OF TABLES Table 2.1 kcat values for the self-malonylation of ACP as measured by KGDH and the KSα/KSβ assays.........56 Table 3.1 Kinetic parameters for the decarboxylation of malonyl-ACP by KSβ (see Appendix 1 for Michaelis-

Menten analysis). .....................................................................................................................................................79 Table 4.1 Kinetic parameters for CHS.170 ..............................................................................................................85 Table 4.2 Kinetic parameters for the reaction catalyzed by AT-KS didomains from modules 3 and 6 using

ACP from every module as a substrate. .................................................................................................................87 Table 4.3 Catalytic constants in for a variety of ACP in the presence of excess acetyl-ACP. ...........................91 Table 5.1 Summary of kcat values for WT and CA KSα/KSβunder a range of conditions. ................................101 Table 6.1 Kinetic parameters for the reduction of unnatural substrates by KR.203...........................................113 Table 8.1 Plasmids transformed into E. coli ........................................................................................................138 Table 8.2 S. coelicolor A3(2) CH999 strains used in this work..........................................................................139 Table 8.3 Primers for site-directed mutagenesis of R72 ACP. Codons containing the mutation R72A are

underlined...............................................................................................................................................................139 Table 8.4 Buffers used for Nickel affinity chromatography ................................................................................146 Table 8.5 Stacking and Separating gels for SDS-PAGE. ....................................................................................146

xix

ABBREVIATIONS

Arg Arginine Asn Asp

Asparagine Aspartate

ACP ACPS

Acyl carrier protein Acyl carrier protein synthase

act ARO

actinorhodin Aromatase

AT Acyltransferase CLF Chain Length Factor CHS CYC Cys

Chalcone synthase Cyclase Cysteine

Da Daltons DH Dehydratase DNA Deoxyribonucleic acid dNTP Dau DTT

Deoxynucleotide triphosphate Daunorubicin-doxorubicin polyketide synthase Dithiothreitol

E. coli Escherichia coli EDTA Ethylenediamine tetraacetate ER ESMS

Enoyl reductase Electrospray mass spectrometry

FAS FPLC

Fatty acid synthase Fast protein liquid chromatography

fren Frenolicin Gln Glutamine Glu Glutamate Gly Glycine gra granaticin His HPLC

Histidine High performance liquid chromatography

IPTG Ile Jad KASI, II and III

Isopropylthio-β-D-galactoside Isoleucine Jadomycin β-ketoacyl:ACP synthases I, II and III

KR Ketoreductase KS Ketosynthase LB Luria-Bertani Medium – media for growth of E. coli Leu Leucine Lys Lysine MCAT Met

Malonyl CoA:ACP transacylase Methionine

NMR Nuclear magnetic resonance OD600 Optical density at 600 nm ORF Open reading frame otc/oxy Oxytetracycline PCR Polymerase chain reaction Phe Phenylalanine PKS Polyketide synthase Pro Proline rpm Revolutions per minute SDS-PAGE Ser

Sodium dodecyl sulphate – polyacrylamide gel electrophoresis Serine

STS Stilbene synthase TCA TCEP

Trichloroacetic acid Tris(2-carboxyethyl)phosphine

tcm Tetracenomycin TE Thioesterase

xx

TFA Thr Tris

Trifluoro acetic acid Threonine Tris(hydroxymethyl)aminoethane

Trp Tryptophan Tyr Val

Tyrosine Valine

Chapter 1. Introduction

1

1. Introduction

1.1. Natural products

The term ‘natural product’ is usually reserved for organic compounds of natural origin that

are unique to an organism or common to closely related organisms.1 In general, the

metabolism of natural products is carried out by specific enzymes, encoded by specific

genes, and the biosynthetic pathways involved are often distinct from the biosynthesis of

essential cell components such as fatty acids, nucleic acids and proteins, which are the

products of the primary metabolism. Thus natural products are biosynthesized by secondary

metabolic pathways that draw upon resources from primary metabolism (i.e. nutrients,

energy). Although natural products may meet the needs of specific individual producers,

they are more often seen as beneficial, or problem-solving, good for the community, be that

of micro-organisms, plants or marine or terrestrial animals (for example, pheromones and

signalling molecules, antifeedants and defense mechanisms, etc.).2

There are four main classes of natural products, according to their biosynthetic origin:

polyketides, isoprenoids, alkaloids and natural products derived from the shikimate

pathway. The latter three types of natural products are beyond the scope of this thesis.

Polyketide biosynthesis is discussed thoroughly in Section 1.2.

1.2. Polyketide biosynthesis

Polyketide natural products are secondary metabolites that are derived from the

condensation of short-chain carboxylic acids. They are produced by a wide variety of

organisms ranging from bacteria to plants, and from fungi to marine organisms. Polyketides

are also important from a pharmaceutical point of view, as many of them exhibit biological

properties such as antibiotic, antifungal, anticancer, cholesterol lowering agent or

immunosuppressant activities (Figure 1.1).


2

O

O

OOH

O OHOH

2

OH

OH

O Cl

O

O

OOO

O

OO

O

OH

O

O

O

O

HO

OOH

O

OO

O

HOH

O

ON

O O OH

O

O

OH O

OH

O

OH NH2

O

O

O OH

OHO

OHOH

OH

O

O

H

Figure 1.1 Polyketides exhibit wide structural variety and pharmacological activities.

1.2.1. Polyketide biosynthesis is similar to fatty acid biosynthesis

Despite the structural variety exhibited by polyketides (Figure 1.1), their biosynthetic

pathways are closely related to one another. Furthermore, the biosynthesis of polyketides is

very similar to that of fatty acids such as palmitate 1, which are products from primary

metabolism.

6-methylsalicylic acid antibiotic

Penicillium patulum

Daunorubicin anticancer

Streptomyces peucetius

Tetracenomycin C antibiotic

Streptomyces glaucescens

Griseofulvin antifungal

Penicillium griseofulvin

Actinorhodin antibacterial

Streptomyces coelicolor (A3)2

Rapamycin inmunosuppresant

Streptomyces hygroscopicus

Lovastatin Cholesterol-lowering agent

Aspergillus terreus


3

HO

O

1

Polyketides and fatty acids are biosynthesized by one or more enzymes that form a

polyketide or fatty acid synthase. These synthases use carboxylic acids as substrates to

make polyketide or fatty acid products, respectively. The key enzyme in polyketide and

fatty acid synthases is a β-ketoacyl synthase (KS). This enzyme catalyzes a carbon-carbon

bond-forming reaction between an acyl thiolester substrate and an acyl group attached to

KS.3,4 This reaction proceeds in two chemical steps (Scheme 1.1): first, the acyl thiolester

substrate 2, which is covalently bound to the active sulphydryl of a protein called acyl

carrier protein (ACP), is decarboxylated. This generates the carbanion 3. The second step is

a Claisen-type condensation between the carbanion 3 and a KS-bound thiolester

intermediate 4 to produce a β-ketothiolester 5. This reaction is repeated several times to

generate an ACP-bound polymer which is extended by two carbon units every

condensation.

An important difference between fatty acid synthases (FAS) and polyketide synthases

(PKS) is the nature of the starter and extender units; i.e. the initial KS-bound thiolester 4

and the acyl thiolester substrate 2. While FAS almost exclusively use acetate and malonate

for chain initiation and extension, respectively, PKS can accept a variety of starter and

extender units. In part, this accounts for the diversity of polyketide structures compared to

fatty acids.

ACP-S O-

O O

ACP-S

O

CO2

ACP-S

O

S-KSR

O

ACP-S

O

KS-S-

O

R!"

Scheme 1.1 Archetypical two-steps mechanism for the generation of a new carbon-carbon bond by FAS and

PKS: 1. Decarboxylation of activated carboxylic acid; 2. Claisen condensation.

3

4 5

2

1.

2.


4

Another important difference arises from the processing of the nascent β-ketothiolester 5.

In fatty acid biosynthesis each condensation is almost always followed by a set of three

sequential reactions at the β-carbon: a ketoreductase (KR) domain reduces the β-keto group

to form a secondary alcohol 6, a dehydratase (DH) promotes dehydration to leave an α,β

double bond 7, and an enoyl reductase (ER) catalyzes reduction of the double bond to

afford a fully saturated thiolester 8 (Scheme 1.2). On the other hand, a PKS may or may not

omit some or all of these β-carbon processing reactions after each condensation, which

results in different reduction levels at each β-carbon. This is the key difference between

FAS and PKS, and is usually known as PKS ‘programming’.5 The length of polyketide

chains (and therefore the number of condensation reactions) and the presence of additional

catalytic domains, such as C-methyltransferases (CMeT) which transfer a methyl unit to the

α-carbon in some bacterial and many fungal polyketide synthases, are further source of

diversity.

ACP-S

O O

R ACP-S

O OH

R ACP-S

O

R ACP-S

O

R!"

KR DH ER

5 6 7 8 Scheme 1.2 Processing of the β-carbon after Claisen condensation in FAS.

The two major protein architectures of FAS and PKS are named Type I and Type II. Type I

enzymes are large multifunctional polypeptides, while type II systems catalyze the

individual reactions using separate, distinct proteins. Type I FAS are found in animals and

fungi, whereas Type II FAS are typical of bacteria and plants. Correspondingly, Type I

PKS are typical of fungi, while bacteria employ Type I and Type II PKS. There is a third

type of PKS, namely Type III PKS, which is typical of plants (and also found in some

bacteria and fungi) and does not have a counterpart in fatty acid biosynthesis.

In the following sections, the enzymology of the different types of PKS is discussed

starting with the most relevant to this thesis, i.e. Type II PKS. Later, Type I PKS in bacteria

and fungi, as well as Type III PKS, will be overviewed by comparison to Type II PKS. As

fatty acid synthases can provide useful information for the characterization of polyketide

synthases, fatty acid biosynthesis will be discussed in parallel with polyketide biosynthesis

when appropriate.


5

1.2.2. Type II polyketide synthases

Actinomycetes are a rich source of bioactive polyketides. Streptomyces species in particular

produce more than one third of all known antibiotics to date.6 Streptomyces employ three

types of PKS organisations; i.e Type I, Type II and Type III PKS.

Bacterial Type II PKS can be defined as complexes of several monofunctional proteins

that convert specific carboxylic acids into polycyclic aromatic polyketides.7 Type II

polyketide synthases are then organised as an assembly of enzymes, brought together by

non-covalent forces, that acts iteratively. This feature as well as the availability of genetic

and molecular tools amenable to actinomycetes prompted the study of aromatic polyketides

as a model for more complicated bacterial or fungal polyketide synthases.

In most cases, the carboxylic acids used as a substrate by Type II PKS are activated as

coenzyme A (CoA) thiolesters. The terminal sulphydryl group in the phosphopantetheine

(PP) arm of CoA is the point to which acyl groups are attached, and then form an acyl-

CoA, such as malonyl-CoA (Figure 1.2).

OH

P

O

O O

N

NN

N

NH2

O

O OH

OP

O

OH

PO

OH

OH

NH

OH

O

NH

S

O

HO

O O

Figure 1.2 Malonyl CoA is an important acyl carrier molecule.

There is some variety in the choice of starter unit for Type II PKS-derived polyketides. For

example, actinorhodin 9,8 jadomycin 10,9 granaticin 1110 and griseusin 1211 are synthesized

from an acetate starter unit. Other Type II PKS employ alternative starter units, such as

propionate (daunorubicin 13,9 doxorubicin 14,9 aclacinomycins12), (iso)butyrate (R1128

1513), malonamate (oxytetracycline 1614) and benzoate (enterocin 1715), (Figure 1.3). On

the other hand, chain elongation is carried out exclusively from malonyl CoA.

Malonate

Phosphopantetheine arm

3’Phosphoadenosine monophosphate


6

These and other natural products formed by Type II PKS comprise an important and

structurally diverse group of secondary metabolites, and many of them (or their

biosynthetic derivatives) have clinically useful anticancer (e.g. daunorubicin, doxorubicin)

and antibiotic (e.g. oxytetracycline) activities. The first Type II PKS-derived polyketide

studied was actinorhodin, whose biosynthesis is reviewed in the next subsection.

Figure 1.3

1.2.2.1. The actinorhodin polyketide synthase

The genome of Streptomyces coelicolor A3(2) was sequenced in 2002. More than 20 gene

clusters were found which corresponded to known or predicted secondary metabolites,

including polyketides, non-ribosomal peptides and terpenes.16 One of them, the polyketide

antibiotic actinorhodin 9 had been known since much earlier (1970s), when the cloning of

bacterial polyketide synthase genes began.

In early work by Rudd and Hopwood, an actinorhodin-producing strain of S. coelicolor

was subjected to random UV mutation. Selection of the mutants that did not produce

actinorhodin was possible due to the absence of the characteristic blue colour of

actinorhodin. A series of 76 S. coelicolor A3(2) mutants were selected in this way.17 These

actinorhodin-blocked mutants were classified into seven classes according to their

phenotype; i.e. the compounds that they produced. For instance, the so-called actIII mutants

failed to secrete any metabolite that could be converted by other mutants to actinorhodin, so

they were deduced to be interrupted in an early step of the actinorhodin biosynthesis.

Whereas the actIII mutants produced a red pigment, the actI mutants were non-pigmented.

Thus, the actI mutants were thought to be incapable of assembling a polyketide, whereas

the actIII mutants would produce a polyketide but would not carry out an essential

modification in the polyketide chain. Moreover, actIII mutants could not convert

compounds secreted by other classes of mutants to actinorhodin. These further mutants

(actIV, VA, Vb, VI, VII) were deduced to be blocked at later stages in actinorhodin

biosynthesis.17

Malpartida and Hopwood then isolated a large (30 Kbp), continuous segment of DNA

from actinorhodin-producing S. coelicolor and showed that this DNA was able to

complement the seven classes of act mutants (actI-actVII) and restore production of

actinorhodin.18 Furthermore, introduction of this DNA on an actinorhodin-sensitive strain


7

of S. parvulus not only reconstituted actinorhodin production but also conferred immunity

to the host.18 The entire actinorhodin gene cluster had been cloned.

The notation of the actinorhodin genes and proteins had been devised according to the

phenotype of the mutants.17 Later, when the actIII region was sequenced, an open reading

frame (ORF) encoding a ketoreductase was found.19 The actI region revealed three open

reading frames encoding three different proteins: ORF1 (ketosynthase, KSα), ORF2 (β

component of the ketosynthase, KSβ) and ORF3 (acyl carrier protein, ACP). ActVII

encoded an aromatase (ARO) and actIV a cyclase (CYC) (Figure 1.4).20

actI

actIII actVII actIV

1 2 3

Figure 1.4 The core of the actinorhodin (act) PKS genes.21

These studies were carried out by means of mutation or deletion in regions of the

actinorhodin PKS cluster, which consists of 23 genes (including structural, regulatory,

export and self-resistance conferring genes). Further understanding of the PKS required a

more convenient strategy. The generation of a derivative strain of S. coelicolor A3(2) that

lacked the entire set of genes of the actinorhodin cluster, namely S. coelicolor A3(2)

CH999, provided an actinorhodin non-producing host for subsequent in vivo studies.22 The

introduction of the desired sets of PKS subunits into this deficient strain of S. coelicolor

allowed the identification of the genes and proteins responsible for the early steps in

actinorhodin biosynthesis (Scheme 1.3).

Three proteins, namely ACP, KSα and KSβ were found to be essential to assemble an

hypothetical octaketide intermediate 18 from malonyl CoA. This set of proteins was termed

the act ‘minimal’ PKS.23 This minimal system was also presumed to catalyze the formation

of the first ring. In the absence of any other enzyme, spontaneous cyclisation and

dehydration of the resulting polyketide intermediate resulted in the isolable SEK4 19.24

Scheme 1.3


8

A ketoreductase (KR, actIII) was found to reduce 18 at C-9 to give an alcohol, and then

mutactin 20 was produced if no other enzymes were present.24 An aromatase (ARO, actVII)

was needed to aromatase the first ring of the polyketide, which spontaneously cyclised to

SEK34 21 in the absence of a cyclase (CYC, actIV).25 ActIV cyclased the second ring to

generate the hypothetic bicyclic intermediate 22.25 In the absence of tailoring enzymes, 3,8-

dihydroxy-1-methylanthraquinone-2-carboxylic acid (DMAC) 23 was produced.25 Further

modifications of the intermediate 22 lead to actinorhodin 9.

Studies have since moved to the area of enzymology and biochemical characterization of

the actinorhodin PKS. Efforts have been made to shed light onto the biochemistry of further

steps in actinorhodin biosynthesis.10,26,27,28,29 In particular, the group of Ichinose has

recently proposed a complete biosynthetic pathway for actinorhodin (Scheme 1.4).28

Following production of the bicyclic intermediate 22, ketoreduction at carbon 3 produces

the hydroxyacid 24, which cyclises and is dehydrated to give the intermediate 4-dihydro-9-

hydroxy-1-methyl-10-oxo-3-H-naphto-[2,3-c]-pyran-3(S)-acetic acid (DNPA) 25.

Oxidation of 25 at carbon 6 affords dehydrokalafungin 26, which is two steps from

actinorhodin: hydroxylation of C-8 of 26, and the dimerization reaction. Valton, Fontecave

and co-workers demonstrated that actVA-ORF5 and actVB catalyze the

monohydroxylation at C-8 of 27 in vitro.26 To date, the enzyme responsible for the

dimerization reaction has not been identified.

During the last decade, much effort has been dedicated to solve the X-ray crystal structure

or NMR structure of the proteins that constitute the act PKS. To date, the NMR solution

structure of the act ACP,30 and crystal structures of ketosynthase KSα/KSβ complex,31 the

KR32 and the monooxygenase enzyme ActVA ORF633 have been published.

This has facilitated the design and interpretation of experiments, as well as the

generation of mutant proteins in order to prove the chemical mechanism of the enzymes.

This is especially true in the case of the act minimal PKS. In vitro studies of the act

minimal PKS have been performed for more than ten years, and the following section

reviews the ‘state of the art’.


9

OOH

OH

O

OO

OOH

OH

O

OHO

OOH

OH

O

OH

O

OOH

OH

O O

OOH

OH

O O

OOH

OH

O O

O

OH

H

O

O

OH

OH

OH

H

O

O

OH

OH

OOHO HO

Scheme 1.4 Proposed later steps in actinorhodin biosynthesis.28

1.2.3. The actinorhodin minimal PKS

As described in Section 1.2.2, early in vivo experiments showed that the act minimal PKS

consisted on an acyl carrier protein (ACP), and two β-ketoacylthiolester synthase

(KSα/KSβ) components.17,20,23 These experiments led to the generation of S. coelicolor

CH999/pSEK4, the strain carrying the genes that encode the act minimal PKS. This strain

was capable of assembling the octaketide SEK4 19.23 SEK4 was assumed to be the product

of a C7-C12 intramolecular aldol condensation of a hypothetical octaketide intermediate 18

(Scheme 1.5). Fu, Khosla and co-workers then described the isolation of a second product

of S. coelicolor CH999/pSEK4, and named it SEK4b.34 SEK4b 27 arises from a C10-C15

cyclisation of 18. In vivo, the ratio 19:27 was approximately 1:1.24

In vitro studies were facilitated by the cloning, expression and purification of ACP and

KSα/KSβ during late 1990s. This greatly contributed to the understanding of the catalytic

properties of each enzyme, which are described in the following subsections. Once the

22 24

25

9

26

ActVI-ORF1

ActVI-ORF1 ?

ActVI-ORF1 + ActVI-ORFA?

ActVI-ORF2

ActVA-ORF6

ActVA-ActVB?


10

catalytic abilities of ACP and KSα/KSβ are described, the various models that have been

proposed for the biosynthesis of SEK4 and SEK4b by the act minimal PKS in vitro will be

discussed.

HO

O O

S-CoA

HO

HO

O

O

OH

O

O

O

OHO

O

OH

HO

O

S-ACP

O O O O O O O O

12

34

56

78

910

1112

1314

1516

7

12

127

1015

Scheme 1.5 Biosynthesis of SEK4 and SEK4b by S. coelicolor CH999/pSEK4, which encodes the act

minimal PKS.

1.2.3.1. The actinorhodin Acyl Carrier Protein (ACP)

Acyl carrier proteins are small (80-100 amino acids), acidic (pI of act ACP = 4.0) proteins

essential to fatty acid and polyketide biosynthesis. In Type I fatty acid and polyketide

synthases, the ACP domain is covalently attached to multifunctional megasynthases, while

Type II systems have catalytic functions in separate proteins, and employ discrete acyl

carrier proteins encoded by distinct genes.

The act ACP was the first PKS protein to be studied structurally.30 Subsequently the ACP

structures of other Type II PKS acyl carrier proteins or Type I and Type II FAS acyl carrier

proteins have been determined.36,37,38,39,40,41 The fold motif of acyl carrier proteins is similar

in most PKS and FAS systems. In general, it consists of three or four helices linked by loop

regions. For example, the structure of act apo-ACP consists of three major helices (I,

comprising residues 7 to 16; II, 42-53; and IV, 72-85), a shorter helix (III, 62-67), a large

18

SEK4 19 SEK4b 27

7-12 cyclization

10-15 cyclization

S. coelicolor CH999/pSEK4


11

first loop separating helices I and II, and a shorter second loop connecting helices II and III

(Figure 1.5). Helices I and IV run antiparallel and are stabilized by numerous contacts

between each other. Helix II runs almost parallel to helix IV. Helices I, II, and IV pack

around a central hydrophobic cleft.30

Figure 1.5 Structure of actinorhodin apo-ACP (PDB file: 2AF8), coloured from N-terminus (blue) to C-

terminus (red).

Inactive, apo-ACP species need be post-translationally modified to afford active, holo acyl

carrier proteins. In vivo, the apo form of ACP is post-translationally modified to holo-ACP

by transfer of 4’-phosphopantetheine (PP) from CoA to a strictly conserved serine residue

located at the bottom of helix II (S42 in act ACP). This reaction is catalysed by a holo-acyl

carrier protein synthase (ACPS), (Scheme 1.6). The PP arm houses an end sulphydryl that

is the attachment point of the acyl groups to holo-ACP. For this reason, holo-ACP is

represented as ACP-SH in biosynthetic schemes throught this thesis.

Heterologous expression and purification of S. coelicolor ACPS by the Bristol group

allowed the study of its catalytic properties in vitro.42 S. coelicolor ACPS is a versatile

phosphopantetheinyl transferase, capable of recognizing a wide range of bacterial FAS and

PKS acyl carrier proteins, including act ACP.

Helix I Helix II

Helix III

Helix IV

Loop I

Loop II


12

OH

P

O

O O

NH

OH

O

N

NN

N

NH2

O

O OH

PO

OH

OH

OP

O

OH

SHNH

O

Scheme 1.6 Act holo-ACP consists of apo-ACP plus a PP cofactor (shown in red) transferred by ACPS from

CoA to Ser42 in ACP.

Acylation of an ACP occurs when a starter or extender unit is attached to the sulphydryl

nucleophilic group at the end of the PP arm. In fatty acid biosynthesis, this reaction is

catalyzed by an enzyme known as malonyl CoA: ACP transacylase (MCAT), (Scheme 1.7).

By analogy to FAS systems, Type II polyketide synthases were expected to utilise discrete

MCAT activity to load the ACP with malonyl units. Since genes encoding MCAT are

absent in most type II PKS genes, it was proposed that FAS MCAT enzymes in

Streptomyces were able to activate both FAS and PKS acyl carrier proteins, in a process

often referred to as ‘cross-talk’ between primary and secondary metabolism.43,44 In this

model, type II PKS systems would recruit MCAT from fatty acid biosynthesis, and the

actinorhodin minimal PKS would involve a fourth component, i.e. S. coelicolor MCAT.

S OH

OO

CoA

S OH

OO

ACP

FASHS+

ACP

FAS

Scheme 1.7 MCAT catalyzes the malonylation of ACP in FAS systems.

1.2.3.2. S. coelicolor malonyl-CoA: ACP transacylase (MCAT)

In S. coelicolor fatty acid metabolism, the corresponding acyl carrier protein (FAS ACP) is

loaded with a malonyl unit in an MCAT-catalyzed reaction (Scheme 1.7). S. coelicolor

ACPS

Apo-ACP Holo-ACP

MCAT


13

MCAT is a 32 KDa monomeric protein. Its crystal structure at 2.0 Å resolution was solved

in 2003 by Stroud and co-workers.45 MCAT is composed of two subdomains (which are

coloured red and blue in Figure 1.6), with the catalytic S97 on a nucleophilic elbow at the

core of a conserved GHSXG motif within the large subdomain.

Figure 1.6 Crystal structure of S. coelicolor MCAT, highlighting the two subdomains (blue and red) and the

catalytic active site S97 (green). PDB code: 1NM2.

The Bristol group studied the malonyl transferase activity of S. coelicolor MCAT in

detail.46 The mechanism and kinetics of the MCAT-catalysed malonylation of S. coelicolor

FAS ACP were investigated. The transfer of malonate from CoA to ACP was found to

operate via a two-step mechanism where MCAT is first acylated by malonate at S97, and

then malonate is transferred to the sulphydryl of the PP arm in holo-ACP (Scheme 1.8).

MCAT

ACP

S OH

OO

CoA+

MCAT

SH

S OH

OO

MCATS OH

OO

+SH

S OH

OO

ACP

CoA

MCAT

Scheme 1.8 Two-step mechanism for MCAT-catalyzed transfer of malonate to FAS-ACP. A. Loading of

malonate onto MCAT; B. Transfer of malonate to holo-ACP and regeneration of MCAT.

A.

B.


14

1.2.3.3. The actinorhodin ketosynthase complex (KSα/KSβ).

In the actinorhodin PKS, polyketide chains are synthesized by a two-component β-

ketoacylthiolester synthase. The two subunits are separate proteins each of ca 45 KDa.

They are usually known as KSα/KSβ or KS-CLF. The former nomenclature will be used

here.

By sequence comparison with other known members of the thiolase superfamily, it

soon became clear that KSα catalyzed the formation of carbon-carbon bonds via Claisen

condensation reactions (Scheme 1.9).20

S

O

thiolase

S

O

R

Substrate carrier

Scheme 1.9 Reaction catalyzed by a general thiolase from a thiolester substrate.48

The most important groups of thiolase enzymes are fatty acid and polyketide β-

ketoacylsynthases (KAS or KS). The active site architecture of thiolases includes a

conserved Cys-His-His (or Cys-His-Asn in Type III PKS) catalytic triad to perform

decarboxylative Claisen condensations.

Rock, White and collaborators have recently proposed a mechanism for the Claisen

condensation catalyzed by KASII of Streptococcus pneumoniae, based on a combination of

structural and biochemical information.49 They generated a series of mutant enzymes

(including mutation of the residues in the catalytic triad, i.e. C169A, H309A and H346A,

act KSα numbering). The catalytic properties of these enzymes, as well as the effect of

mutations in protein structure and ACP binding, were assessed.

For instance, while C169 and H346 were essential for condensation, the H309A mutant

retained some activity. Comparison of the crystal structures of the wild type (WT) and

H309A S. pneumoniae KASII led to the observation that there was a water molecule in the

active site of the WT protein, which the H309A mutant lacked. This water molecule was

hydrogen-bonded to the Nε of H309.


15

In their model, H309 is the anchor that positions a catalytic water molecule W1 in exactly

the right place to perform a nucleophilic attack on the carboxylate 28 of the substrate and

release bicarbonate (Scheme 1.10). There was evidence that bicarbonate was the product of

the decarboxylation reaction, rather than CO2, because bicarbonate inhibited malonyl-ACP

decarboxylation. His346 promotes the formation of the carbanion 29 at C2 of the malonate

by stabilizing the enolate intermediate. The carbanion reacts with the acyl-Cys169

intermediate, and the tetrahedral transition state resolves to form the β-ketoacyl-ACP

product.

NH

O

ACP-S

O

OH

N

HN

His309His346

O

H

H

N

NH

O

ACP-S

O

OH

N

HN

His309His346

H

HO

O

R S

Cys169

HN

N

Scheme 1.10 Mechanism of two-step decarboxylative Claisen condensation in thiolases (act KSα

numbering), proposed.49

The structure of act KSα resembles that of the thiolase superfamily fold. KSα has two

halves, the N-terminal half and the C-terminal half, which have a βαβαβαββ secondary

structure. The two halves are assembled in a five layered α-β-α-β-α bundle structure

(Figure 1.7). The essential and fully conserved cysteine (C169) active site is in the Nβ3-

Nα3 loop, in a position in which the electrostatics of helix Nα3 lower the pKa of the

reactive cysteine, making it more nucleophilic.50,51

1.

2.

W1

28

29


16

Figure 1.7 Structure of KSα, showing from top to bottom the typical five layered α-β-α-β-α bundle fold of

thiolase enzymes. The active site cysteine is highlighted. PDB file: 1TQY

Most enzymes of the thiolase superfamily are dimers, tetramers or hexamers, and

monomers have not been observed.48 Thus before direct experimental evidence had been

obtained, it was accepted that KSα and KSβ formed at least a heterodimer. While the role

of ACP and KSα became apparent ever since their sequence were known, the function of

KSβ was unclear.20 KSα and KSβ are very similar to each other (40% similarity, 26%

identity) nevertheless a fundamental difference between the proteins is that the KSα active

site has an essential cysteine (Cys, C) active site, whereas in the KSβ the corresponding

residue is a highly conserved glutamine (Gln, Q). This glutamine is conserved along KSβ

from many Type II PKS as well as the loading domain (KQ) on Type I modular PKS such

as niddamycin (nid) and tylosin (tyl) (Figure 1.8).

Early in vivo experiments by McDaniel, Khosla et al provided the first insights into the

properties of KSβ. The KSα, KSβ and ACP from the octaketides actinorhodin 9 and

granaticin 10 PKS, and the decaketide tetracenomycin (tcm) 30 polyketide synthases were

combined. The chain length of the polyketides produced was then analyzed. It appeared

that KSβ was the main determinant of chain length because, when tcm KSβ was coupled

with act KSα, a decaketide was produced. Conversely, when tcm KSα was paired with gra

KSβ, an octaketide was detected.22 The Stanford group hence baptized KSβ ‘chain length

factor’ (CLF).

α bundle

β bundle

α bundle β bundle

α bundle


17

Figure 1.8 Primary sequence alignment of the active site (green star) region of a range of FAS ketosynthases

as well as KSα and KSβ from various PKS. For a complete sequence alignment see Appendix II.

OH

CH3OHO

O

O

O

OHOH

OO

O

30

1.2.3.4. The Stanford model for the act minimal polyketide synthase in vitro

Within the Stanford group, Carreras and Khosla first reconstituted PKS activity using act

KSα/KSβ, holo-acyl carrier proteins from a variety of Type II polyketide synthases (but not

act ACP), and MCAT.53 In the absence of MCAT, no activity was detected. This result

apparently supported the idea of a ‘cross-talk’ between fatty acid and polyketide

biosynthesis in S. coelicolor,43,44 because an enzyme from the primary metabolism was

necessary to enable production of polyketides in vitro.

Therefore, in the Stanford model, the formation of malonyl-ACP from holo-ACP and

malonyl CoA is catalyzed by MCAT (Scheme 1.11.A). Initiation of polyketide synthesis

proceeds via decarboxylation of malonyl-ACP to acetyl-ACP, catalyzed by KSα. The

acetyl unit generated is transferred to the active site cysteine in KSα (Scheme 1.11.B).

Iterative loading and Claisen condensation steps lead to elongation of the chain (Scheme

1.11.C). The elongating chain extrudes into a ‘polyketide tunnel’ created between KSα and


18

KSβ (Scheme 1.11.D). When the end of the tunnel is reached, the chain buckles, and

cyclization and release occur.

ACPS OH

OO

CoA+SH

S OH

OO

ACP

CoA

MCAT

S

HS

KS! KS"

HO

O O

CO2

S

HS

O

ACP ACP

KS! KS"

S

S

HO

O O

CO2

S

HS

O

ACP ACP

O

R

O

R

KS! KS" KS! KS"

S

OO

ACP

O O O O O O

Scheme 1.11 The Stanford model for production of SEK4 and SEK4b by the act minimal PKS.31 A. Loading

of ACP is catalyzed by MCAT. B. Decarboxylation of malonyl-ACP is catalyzed by KSα, which

subsequently takes the acetyl starter unit. C. Claisen condensation and elongation of the polyketide chain. D.

The polyketide chain extrudes in a tunnel formed in the interface between KSα and KSβ, and finally cyclizes

and it is released from the PKS.

In remarkable work, the Stanford group identified two key residues in KSβ that help form

the polyketide tunnel.54 Two phenylalanines (F109 and F116) in KSβ were mutated to

alanines, and the corresponding single and double mutant KSα/KSβ complexes were

purified. These mutations introduced a less bulky aminoacid in the position of two aromatic

residues; thus they aimed to increase the chain length of the polyketides produced. In vitro

assays with purified ACP, MCAT and KSα/KSβ complexes showed that the mutant F116A

KSβ

KSα

SEK4 + SEK4b Cyclization and release

A.

B.

C.

D.


19

produced a ratio 64:36 of decaketides to octaketides (as determined by HPLC analysis),

while the F109A/F116A double mutant synthesized decaketides as >95% of total

polyketides.54

1.2.3.5. The Bristol model for the actinorhodin minimal polyketide synthase in vitro

The Bristol group was able to reconstitute PKS activity in vitro using only holo-ACP and

KSα/KSβ, thus in the absence of MCAT.8 Indeed, the loading of holo-ACP with malonate

from malonyl CoA to form malonyl-ACP was shown to be catalyzed by holo-ACP itself

(Scheme 1.12).55 This ‘self-acylation’ activity of holo-ACP from the actinorhodin 9,

griseusin 12 and oxytetracycline 16 PKS was proven with a range of substrates, CoA and

SNAC derivatives.55

CoA-SHS OH

OO

ACPHS

CoA

S OH

OO

ACP

Scheme 1.12 Self-malonylation of holo-ACP

Awkwardly, the debate about the capability of Type II acyl carrier proteins to catalyse their

own acylation was continuous in the literature during the following years. The Stanford

group could not reconstitute PKS activity using only KSα/KSβ and ACP, and argued that

the reported self-malonylation of ACP was attributed to contamination of ACP preparations

with E. coli MCAT due to the use of E. coli-based over-expression systems.53,56

Reynolds and collaborators observed the self-malonylation ability of the

tetracenomycin and frenolicin acyl carrier proteins.57 Moreover, they observed that these

PKS acyl carrier proteins were able to catalyze the malonylation of FAS ACP, which

suggested that PKS ACP and MCAT could catalyze the same chemical reactions. However,

these reports were later retracted as an artefact of heterologous expression and purification

of ACP from E. coli.58 This was based on two facts: first, that consecutive purification steps

of ACP preparations apparently reduced the self-malonylation activity of the


20

tetracenomycin ACP. Second, that an ACP purified from insect cells (which lack discrete

MCAT activity) was unable to self-malonylate.58

Conclusive evidence in support of the ability of, at least, act ACP to self-malonylate was

gathered when the Bristol group used a chemically synthesized ACP. The self-malonylation

activity of this synthetic ACP unequivocally proved the inherent self-malonylation activity

of act ACP, as such protein could not be contaminated with E. coli MCAT.59

Furthermore, the initial observation by the group of Reynolds that PKS acyl carrier

proteins can catalyze the malonylation of FAS ACP, was also proved true by the Bristol

group. Mass spectrometric studies showed that act and otc malonyl-ACP could transfer

their malonyl groups to a recipient PKS or FAS holo-ACP in vitro.60

Recently, the self-malonylation activity of other Type II ACP (i.e. FAS acyl carrier proteins

from Plasmodium falciparum and Brassica napus) has been reported.61 This is striking

because these FAS systems also employ distinct MCAT activities to catalyze malonylation

of ACP. It is possible that MCAT could ensure the specificity of ACP malonylation

permitting only malonyl CoA to be utilized as substrate.

In the model proposed by the Bristol group for the act minimal PKS,60 the minimal PKS

consists of the KSα/KSβ heterodimer and two molecules of holo-ACP, one of which is

complexed with KSα/KSβ (ACP1) and the other is free in solution (ACP2). In a first step,

the ACP in solution undergoes self-malonylation and loads itself with malonate from

malonyl CoA (Scheme 1.13).60 ACP2 then transfers its malonate to the active thiol of the

complexed ACP1. Malonyl-ACP1 is decarboxylated by KSβ (see Chapter 3) to generate

acetyl-ACP,62 which in turn primes KSα with an acetyl starter unit. Meanwhile, ACP2 has

undergone self-malonylation again, and transferred malonate to ACP1. Then a Claisen

condensation catalysed by KSα generates the first polyketide intermediate. This is

transferred to KSα, and ACP1 is ready to receive an extender unit once more. Six further

condensations need be carried out to produce the first isolable polyketides, SEK4 and

SEK4b. This model describes an associative, self-sufficient minimal Type II PKS. Scheme 1.13


21

1.2.4. Type III polyketide synthases

Polyketides produced by Type III PKS recently received much attention due to their

biological activities, which include antioxidant, anti-inflammatory and anti-cancer

properties.3,63 Type III polyketide synthases are typical of plants, but have also been found

in bacteria and fungi.

The most remarkable feature of Type III PKS is that all the catalytic activities needed

for making the final product are contained within a single enzyme. Indeed, selection of

starter and extender units, decarboxylation of substrates, carbon-carbon bond formation and

processing of polyketides (cyclisation) occur in the same active site of the enzyme. Thus,

Type III PKS possess β-ketoacyl synthase (KS) activity, as well as the ability to cyclise and

release the polyketide, but there is no requirement for any further processing step (e.g.

reduction, elimination). Type III PKS also lack an ACP domain, and starter and extender

units are activated as CoA thiolesters, instead of ACP thiolesters. There are only a few

examples of Type III PKS that accept ACP-bound starter units (e.g. Type III PKS

SCO7221 and SCO7671 from S. coelicolor).64

The architecture of the active site in Type III PKS is very similar to that in KSα

components of Type II PKS.65 The universal cysteine is maintained, as well as the

corresponding histidine to H309 (act KSα numbering). However, in Type III PKS an

asparagine residue substitutes for the second histidine (H346).

The chalcone and stilbene plant polyketide synthases are the best studied Type III

PKS.4 The X-ray structures of chalcone synthase from alfalfa67 and stilbene synthase from

pine68 and peanut69 have been published. These show that type III PKS are small (40-45

KDa), homodimeric enzymes which usually share a high degree of similarity in primary

sequence (50-90% sequence identity within plant PKS, and 25% within bacterial PKS).

The stilbene and chalcone PKS both use p-coumaryl CoA 31 as starter unit and extend it

with three repetitive, decarboxylative condensations with acetate extender units derived

from malonyl CoA (Scheme 1.14). A most remarkable difference between the two enzymes

is the cyclisation of the final polyketide intermediate, which results in the production of two

different polyketides, chalcone 32 and resveratrol 33. The mechanism of this cyclisation is

not fully understood;3,68,69 in fact, despite the high degree of similarity between the two


22

enzymes, the group of Noel needed to mutate eighteen residues in order to convert a

chalcone synthase into a stilbene synthase.68

HO

S-CoA

O

CoA-S

O O

OH

HO

O O

S-Enz

HO

O O O

O S-Enz

HO

OH

HO

OH

O

OH

HO

OH

O

HO

O

O

O

Enz-S

HO

O

O

OO

Enz-S

CO2

Scheme 1.14 Biosynthesis of chalcone and resveratrol (a stilbene) by chalcone (CHS) and stilbene (STS)

synthases.65

1.2.5. Type I modular polyketide synthases

Type I modular PKS, usually found in bacteria such as actinomycetes, are responsible for

the biosynthesis of macrolide compounds such as niddamycin 34 from Streptomyces

caelestis and tylosin 35 from Streptomyces fradiae and polyethers such as monensin 36

from Streptomyces cinnamonensis. In this type of PKS, several large proteins, each

containing more than one catalytic active site, combine in a processive fashion to produce a

polyketide. Each protein is subdivided in subunits called ‘modules’, which are covalently

bound to each other. Each module possesses the required catalytic domains for a round of

chain extension. A classic example of this modular Type I PKS is that of 6-

deoxyerythronolide B (6-dEB) synthase (DEBS) from Saccharopolyspora

erythraea.4,70,71,72,73

33

32

31

Type III PKS

2 x malonyl CoA Type III PKS

CHS

STS


23

O

O

O

O

CH3

H3C

O

O

CH3

OH

OO

HON(CH2)2

OH

O

OH

O

O

34

O

O

O

O

O

CH3

OH

OO

HON(CH2)2

OH

O

OH

OH

OO

OMeOMe

HO

35

OO

HO

MeO O O

O

HO

OHO

HO 36

1.2.5.1. The 6-deoxyerythronolide B (6-dEB) polyketide synthase.

6-dEB 37 is the polyketide part of the commercially used antibiotic erythromycin A 38.

The DEBS consists of three proteins (denoted DEBS1, -2 and -3), each of which is

organised in two PKS modules. Each module contains the necessary domains for a round of

chain extension and modification. In every module, at least an acyltransferase (AT), acyl

carrier protein (ACP) and ketosynthase (KS) domains are present, as well as the necessary

active sites for the modification of the β-keto groups in each round (KR, DH and ER). In

addition, DEBS1 and DEBS3 contain the catalytic domains capable of selecting an

appropriate starter unit (loading) and releasing the final product (thiolesterase, TE),

respectively.

The primary sequence of each module is organised in the same fashion (Figure 1.9).

Linker regions between domains and between modules within the same enzyme are

covalent. Interactions between subunits (between DEBS1 and DEBS2, and between

DEBS2 and DEBS3) occur between the ACP of the previous enzyme and the KS of the

following enzyme. This means that biosynthetic intermediates probably undergo direct


24

transthiolesterification between ACP and KS and are not released into solution. Thus KS

both accepts the growing polyketide chain from the previous module and catalyzes the

subsequent decarboxylative condensation between this substrate and an ACP-bound

methylmalonyl extender unit, introduced by AT.

Figure 1.9 Gen sequence of a DEBS PKS module. KS = ketosynthase, AT = acyl transferase, DH =

dehydratase, ER = enoyl reductase, KR = ketoreductase, ACP = acyl carrier protein. DH and ER are only

present in module 4.

The first of the DEBS proteins contains the loading and two PKS modules. A propionyl

starter unit is loaded onto the ACP by the dedicated AT domain, Scheme 1.15. Module 1

then catalyses the decarboxylation of a methylmalonyl CoA and condensation with the

starter propionyl unit in the KS active site. Reduction of the β-carbonyl by KR yields the

first ACP-bound polyketide intermediate, 3-hydroxy-2-methylpentanoate 39. The second

module is analogous to the first module and contains the same catalytic domains.

DEBS2 contains modules 3 and 4. The third module elongates the polyketide chain

with a new methylmalonyl CoA. Although it carries a ketoreductase domain (KRi, Scheme

1.15), this appears to be inactive. In the fourth module, reduction of the β-carbonyl by KR

is followed by dehydration (DH) and reduction of the double bond (ER) to afford a fully

saturated thiolester.

DEBS3 houses module 5, which elongates the chain with a further methylmalonyl unit

and reduces the corresponding ketone. Module 6 is analogous to module 5. Finally, the

extra TE domain cleaves the thiolester between the polyketide and the ACP, leading to

deoxyerythronolide B 37.

Scheme 1.15

1.2.6. Type I iterative polyketide synthases

Type I iterative polyketide synthases are only found in fungi. Although fungi may use for

example Type III polyketide synthases, most polyketide synthases found in fungi are

KS AT (DH) (ER) KR ACP


25

multifunctional enzymes. These megasynthases (> 200 KDa) house the several catalytic

activities needed for the assembly of polyketides.

As described in Section 1.2.5, each module of a Type I modular PKS acts only once

during polyketide synthesis. Conversely, PKS activity in fungi is iterative, much more like

Type II PKS. Thus, the same multifunctional enzyme acts once and again in successive

rounds of chain extension. In Type II polyketide synthases, ACP and KSα probably build

up the full-length polyketide prior to any tailoring step such as reduction and cyclization

(Scheme 1.11). On the other hand, a fungal PKS may or may not process the nascent β-

carbon after each condensation, leading to different levels of reduction after each round

(see Scheme 1.2 in Section 1.2.1).

As a result, fungal polyketides show huge structural variety. Orsellinic acid 40 from

Aspergillus and Penicillium genera, tetrahydroxynaphthalene 41 from Colletotrichum

lagenarium and norsolorinic acid 42 produced by many Aspergilli are examples of the

simple, non-reduced fungal polyketides; whereas fusarin C 43 from Fusarium species and

tenellin 44 from Beauveria bassiana represent more complex polyketide structures fused to

amino acid derivatives.

OHO

HO

OH

OH OH

HO OH 40 41

OH

HO

O

O

OH O

OH

O

N

OH

O

OH

HO

42 43

OMeO

O

NH

O

O

OH

HO 44


26

1.3. Structure of the mammalian fatty acid synthase as a model for polyketide

synthases.

The similarity between fatty acid and polyketide biosynthesis has been emphasized above.

FAS and PKS share common biochemical and structural features, and insights into Type I

FAS structures have important implications for understanding polyketide synthases.74 Ever

since the study of the biochemistry of fatty acids started 50 years ago, scientists have

speculated about FAS structure and mechanism.

1.3.1.1. Initial experiments: the linear model (1970s-1990s)

Type I FAS and PKS are multifunctional proteins with individual functional domains.

Initially, fatty acid synthases were thought to be large, linear structures75 but it soon (i.e.

1975) became clear that only the dimer form of the synthase was functional.76 During the

decades of 1980 and 1990, first inhibition studies77 and then low resolution electron

microscopy78 suggested that the two linear polypeptides were arranged in a head-to-tail

fashion. In this model, the ACP of one subunit would interact with the KS of the other

subunit (Figure 1.10).

ACP TEERMATKS DH KR

ACPTE ER MAT KSDHKR

Figure 1.10 Head-to-tail model and linear arrangement of a dimeric FAS. Single headed arrows indicate the

order of domains in the primary structure (N-terminus to C-terminus). Blue, double headed arrows indicate

the interaction between ACP and KS domains.

1.3.1.2. Building of a 2D model (1990s)

New experimental data obtained by Joshi, Smith, Witkowski and collaborators during the

late 1990s urged a revision of the linear model. They found that mutant FAS lacking


27

functional KS, DH, ACP or TE domains in both subunits of the dimer (i.e. homodimers), as

well as heterodimers formed between mutant ACP and DH, were unable to synthesize fatty

acids. However, heterodimers formed between mutant KS and DH or ACP regained partial

FAS activity (Figure 1.11).79,80 Thus, the linear model for the animal FAS had to be revised

to reflect the finding that the two constituent polypeptides were not simply positioned side-

by-side in a fully extended conformation, but were coiled in a manner that allowed, for

example, the KS and the DH domains of one polypeptide to access the ACP domain located

thousands of residues away on the same polypeptide. This implied the necessity to account

for head-to-tail contacts both inter and intrasubunits.

Direct experimental evidence in support of the new model was obtained by

dibromopropanone cross-linking experiments. This showed that the PP arm in ACP could

interact with the cysteine in KS of both subunits.82 In the subsequent new proposed model,

the two polypeptide chains are intertwined head-to-tail and head-to-head thus facilitating

the interaction of the ACP of both monomers with the catalytic active sites situated at both

sides of the dimer, especially the MAT and the KS domains (Figure 1.12).79,83,84

ACP TEERMATKS DH KR

ACPTE ER MAT KSDHKR X

X ACP TEERMATKS DH KR

ACPTE ER MAT KSDHKR XX

ACP TEERMATKS DH KR



ACPTE ER MAT KSDHKRX X

X

ACP TEERMATKS DH KR




X

Figure 1.11 Complementation studies with mutant FAS. The inactivated domain is shown by X. A. Active

condensation intersubunit; B. Active condensation intrasubunit; C. Active acyl transfer intersubunit; D.

Active acyl transfer intrasubunit; E. No dehydration intersubunit; F. Active dehydration intrasubunit.81

A.

C.

B.

D.

E. F.


28

ACP

TE

ER

ACP

TE

ER

KS

MATDH

KR

KS

MAT

DH

KR

Figure 1.12 2-D model for the topology of Type I mammalian FAS. Single headed arrows indicate the order

of domains in primary sequence from N-terminus to C-terminus. Blue, double headed arrows indicate

interaction between ACP and KS, and between ACP and MAT.84

1.3.1.3. 3D models for FAS and PKS megasynthases (1990s-2000s)

The publication of the high-resolution (4.5 Å) X-ray structure of the porcine FAS by Maier,

Jenni and Ban was a breakthrough.85 The enzyme was revealed as an iterative

nanomachine, with overall dimensions of 210 Å x 180 Å x 90 Å that are in good agreement

with previous low-resolution electron microscopy observations (Figure 1.13).86 The

positioning of domains revealed the complex architecture of the multienzyme, which forms

an intertwined dimer with two lateral semicircular reaction chambers. Each monomer

contains a full set of catalytic domains required for fatty acid elongation. The complex

adopts an X-shape with a central body extended at the upper and lower ends by so-called

‘arms’ and ‘legs’ (Figure 1.13). The lower part is comprised by the homodimeric KS

domains, and AT forms the legs. This lower body is connected at the waist with an upper

part formed by the DH, ER (upper body), and KR domains (arms). Distances between the

active sites of catalytic domains are in the nm order which means that the ACP possesses an

extraordinary inherent flexibility to perform its function of acyl (i.e. starter and extender

units and intermediates) carrier protein. In fact, neither the ACP or the TE domains could

be assigned, presumably due to their high mobility.


29

Figure 1.13 Structure of Type I FAS megasynthase (PDB file: 2CF2). Yellow, AT; red & green, the two

monomers of KS; blue, DH; magenta, homodimeric ER; orange, KR. The proposed reaction chambers are

marked with a star.

Within polyketide synthases, Type I iterative PKS in fungi are most similar to mammalian

FAS. Indeed, it has been proposed that the structure of Type I iterative PKS resembles that

of the mammalian FAS,5 although no experimental evidence has been obtained to date.

On the other hand, much work has been done with Type I modular PKS, especially with

DEBS. For example, effort has been drawn to elucidate X-ray and NMR structures of

DEBS domains, di-domains and linker regions. To date, the X-ray structures of the KS-AT

di-domain of module 5,87 the KR domain from module 1,88 the TE domain,89,90 the NMR

solution structure of the linker region between DEBS2 and 391 and, more recently, the

solution structure of the ACP from module 292 have been published. By combining all this

information with structural knowledge about Type I and Type II FAS, as well as Type II

71 Å

72 Å

37 Å 32 Å


30

PKS, it has recently been proposed that each module adopts a homodimeric X-shape much

as that of the animal fatty acid synthase.73 This is especially useful for module 4, which

shares with FAS all the reductive domains.

1.4. Fundamentals of enzyme kinetics

All enzymatic kinetic research is based ultimately on the experimental determination of the

catalytic activity of the enzymes. A kinetic test is reliable if (a) the signal is directly

proportional to the variation of the compound measured, (b) the monitoring of reaction

signal is in convenient time range for determining the initial rate unambiguously, and (c)

the reaction rate is linear with respect to enzyme concentration.93 A wide range of

conditions (e.g. enzyme and substrate concentrations, temperature, pH, etc.) must be

assayed in order to characterise the kinetic behaviour of the enzymes.94

A discontinuous assay requires the removal of samples from the reaction mixture at time

intervals and subsequent analysis to determine the extent of reaction, for instance, by

HPLC. Conversely, a continuous assay monitors the progress of the reaction continuously

with an automatic recording device. For example, a well known spectrophotometric effect

in biochemistry is the change in the absorbance at 340 nm when the reduced form of

NAD(P) is oxidised. Changes in fluorescence is another physical property the experimenter

can take advantage of. A change in the pH of the reaction can also be followed and related

to enzyme activity. Even if no spectroscopic or pH change takes place, a coupled enzymatic

assay can often be devised. Here, the reaction of interest is coupled with another reaction,

faster than the target reaction, which does cause a measurable change in some of the

properties outlined above.94

1.4.1. Rate equations in enzyme kinetics

The theory of enzyme kinetics was developed at the beginning of the last century. First

Henri, and then Michaelis and Menten proposed a mechanism where the substrate bound to


31

the enzyme to generate an enzyme-substrate complex, which then released the product and

regenerated the enzyme (Scheme 1.16).94

E + S ES E + P

Kd k2

Scheme 1.16 Mechanism for enzyme-catalyzed reaction proposed by Michaelis and Menten. S = substrate, E

= enzyme, P = product.

Michaelis and Menten then carried out a mass balance assuming that the reversible step was

a true equilibrium, and the concentration of the intermediate ES was simply calculated

through the dissociation constant, Kd. The rate equation derived by Michaelis and Menten

was:

!

rate =k

2E

0[ ] S[ ]Kd + S[ ]

(Eq. 1)

where E0 is the initial enzyme concentration.

Later, Briggs and Haldane examined a more general mechanism in which the reversible

step was not treated as an equilibrium (Scheme 1.17).94

E + S ES E + P

k-1

k2k1

E0-x xS P

Scheme 1.17 Briggs-Haldane mechanism. The concentration of the different species is shown underneath.

The concentration of the intermediate, x, was assumed to reach a constant, steady state

value (Eq. 2).

!

dx

dt= k1( E0 " x )S " k"1x " k2x = 0 (Eq. 2)


32

Collecting terms in x and calculating the rate as k2x gives Eq. 3.

!

rate =k

2E

0[ ] S[ ]k

-1 +k2

k1

+ S[ ] (Eq. 3)

Eq. 3 was derived for a particular mechanism (Scheme 1.17), but it applies to much more

complex systems than the simplest two-steps Michaelis-Menten or Briggs-Haldane

mechanism (Scheme 1.16). It usually takes the general form:

!

rate =kcat

appE0[ ] S[ ]

KM

app+ S[ ]

(Eq. 4)

where kcatapp and KM

app are not true first order rate constant and equilibrium

constants, but the apparent catalytic constant and Michaelis constant, respectively.

If the concentration of substrate is much higher than KMapp, the equation of the rate is as

follows:

!

rate " kcat

appE[ ] (Eq. 5)

Under these circumstances, the rate is the maximum reaction rate achievable at a certain

enzyme concentration and is generally referred to as Vmax. In turn, KMapp can be defined as

the substrate concentration that results in a reaction rate which is half the value of Vmax.

Similarly, at low substrate concentrations (much lower than KM) the quantity kcat / KM is

the second-order reaction rate constant:

!

rate " kcat

/KM

[E][S] (Eq. 6)

kcat / KM is usually referred to as specificity constant because it determines the ratio of rates

for two competing substrates A and B when they are mixed together:


33

!

rateA

rateB

"(V/K

M)S

A

(V"/K"M

)SB

=(k

cat/K

M)S

A

(kcat

"/K"M

)SB

(Eq. 7)

where SA and SB are the concentrations of A and B respectively.

The value of kcat / KM is limited by the diffusion-controlled encounter of the enzyme-

substrate, which is between 108 and 109 M-1 s-1.95 Some of the fastest enzymes known, for

example carbonic anhydrase, triose phosphate isomerase and acetylcholinesterase have kcat /

KM values in that range.

A final, important consideration is that in deriving the rate equations described in this

section, the concentration of the substrate, S, was not corrected to account for the formation

of the intermediate ES or product. This is an essential condition of the Michaelis-Menten

and Briggs-Haldane approaches: the substrate concentration was treated as constant and

equal to the initial value. Consequently, the rate that appears in Eq. 4 is the initial rate, i.e.

the rate of the enzymatic reaction when the substrate concentration either a) is much higher

than KM (>5-10 fold) or b) does not vary considerably (ideally less than 10%) during the

length of the experiment.

1.4.2. Kinetics of the act minimal PKS

The actinorhodin Type II PKS from Streptomyces coelicolor A3(2) has been the model for

Type II PKS in the literature. The act minimal PKS has been extensively studied by the

Bristol and Stanford groups during the last decade.

The act minimal PKS catalyses a number of individual reactions. Firstly malonate must

be transferred from CoA to holo-ACP (Scheme 1.18.A). In the Bristol model for the act

minimal PKS, this loading reaction is catalyzed by ACP itself (Section 1.2.3.5). The

kinetics of self-malonylation of holo-ACP were studied by means of a radioactive,

discontinuous method to measure the transfer of 14C labelled malonate units.55 A catalytic

constant, kcat = 0.34 min-1 and Michaelis constant KM = 219 µM were measured. Malonyl


34

transfer between acyl carrier proteins has also been studied in the Bristol group by

discontinuous mass spectrometry methods, though no kinetic parameters were calculated.

Initiation of polyketide synthesis proceeds via decarboxylation of malonyl-ACP to form

acetyl-ACP, the starter unit for polyketide biosynthesis (Scheme 1.18.B).62 The polyketide

chain is then extended to 16 carbon atoms by seven extension reactions using malonyl-ACP

(Scheme 1.18.C). These reactions are catalyzed by iterative cycles of KSα.96 It has been

traditionally assumed that the polyketide chain is fully formed before the chain cyclizes and

it is released from the synthase to form SEK4 19 and SEK4b 27 (Scheme 1.18.D).

HO

O O

S-CoA

holo-ACP

CoA-SH

HO

O O

S-ACP

CO2

O

S-ACP

x 7 x 1

O O O O O O O

S-ACP

O

OH

H

COOHO

O

OH

OH

OH

H

COOHO

O

OH

OH

HO

HO

O

O

OH

O

O

O

O

HO O

OH

HO

O

Scheme 1.18 Biosynthesis of octaketides by the act minimal PKS. A. Loading of holo-ACP with malonate. B.

Initiation of polyketide synthesis by formation of acetyl-ACP. C. Elongation of the polyketide chain. D.

Formation of SEK4 and SEK4b in vitro. E. Further tailoring reactions lead to actinorhodin in vivo.97

Remarkably, little is known of the absolute or relative rates of the component reactions of

the act minimal PKS, although the overall transformation from malonyl CoA to SEK4 and

SEK4b has been studied. The Stanford group measured SEK4 and SEK4b production in

MCAT-supplemented act minimal PKS.56 A discontinuous thin layer chromatography

based method for detection of 14C labelled SEK4/4b was used. Under their experimental

conditions (and using an ACP from the frenolicin PKS), a KM of 5 µM for holo-ACP was

given as well as a catalytic constant kcat of 0.31 ± 0.11 min-1. Subsequent publications from

19

27 9

A. Loading B. Initiation

C. Extension

D. In vitro: cyclisation and release

E. In vivo: act PKS


35

the same group have reported similar kinetic parameters, in the range kcat = 0.11-0.27 min-1

and KM = 2.7-6.4 µM with ACP from a variety of Type II PKS, including act ACP.98,99

However, the ACP used in these studies were not able to load themselves with malonate

units and questions have been arisen towards the quality of these proteins due to the

absence of ACP characterization (e.g. levels of folded, active holo-ACP).60 In any event,

the reaction rates of the initiation and extension steps could not be disclosed.

Within the Bristol group, Matharu performed actinorhodin minimal PKS assays and

measured the initial rate of SEK4/4b production. She varied the concentration of ACP from

2.5 to 200 µM, with constant malonyl-CoA (1 mM) and KSα/KSβ (1.5 µM) concentrations.8

A discontinuous assay was used to follow the reaction: at time intervals, a sample was

taken from the reaction mixture and analysed by HPLC. It appeared that production of

SEK4 and SEK4b occurred at the rate predicted by Hitchman55 for the self-malonylation of

act holo-ACP.8 Therefore, it was hypothesized that the self-malonylation of ACP was the

rate limiting step in the overall production of octaketides by the minimal PKS, even at the

highest ACP concentrations used in the study (200 µM holo-ACP).

1.5. Aim of the project

The aim of this work was to further understand the process of polyketide biosynthesis using

the S. coelicolor Type II actinorhodin PKS as a model system. The first objective of the

project was to develop a method amenable for kinetic purposes to measure the rate of the

reactions catalyzed by each component of the actinorhodin minimal PKS. In this thesis,

Chapters 2, 3 and 4 are devoted to the study of the catalysis of ACP (loading step), KSβ

(initiation step) and KSα (extension step), (Scheme 1.18). Subsequently, the second

objective was to generate a series of ACP, KSα and KSβ mutant proteins and subject them

to these assays in order to identify specific residues responsible for the catalysis of each

protein.

In parallel, we aimed to use a kinetic approach to investigate the stoichiometry of the

act minimal PKS (Chapter 5), and the effect of the addition of further act proteins to the

minimal system (Chapter 6).

Chapter 3. Initiation of polyketide synthesis

36

2. Studies on Acyl Carrier Proteins

Acyl carrier proteins are essential components of Type I and Type II polyketide and fatty

acid synthases. In vivo, acyl carrier proteins are expressed as inactive apo forms, and need

to be postranslationally modified to the active holo forms by addition of a

4’phosphopantetheine (PP) cofactor with its terminal sulphydryl (Section 1.2.3.1).

Previous studies within the Bristol group had shown the rapid disulphide formation

between the thiols of the PP of holo-ACP to form ACP dimers in solutions lacking a

sulphydryl reducing agent (Scheme 2.1).8,52 The preservation of the reactive sulphydryls of

ACP is critical to the maintenance of their function, as the malonate units required for

initiation and extension of the polyketide chain are attached to the sulphydryl at the end of

the PP arm.

Another reaction that leads to inactive ACP forms, at least in vitro, is an intramolecular

disulfide formation between C17 in act ACP and the terminal thiol of the PP arm. For this

reason, a mutant C17S act ACP was engineered within the Bristol group.52 In this thesis,

this C17S ACP will be referred to as the ‘reference’ act ACP, because it was used

throughout our work instead of the wild type act ACP.

ACP

SHACP

SHACP

S S

ACP

Scheme 2.1 Disulphide formation between the thiols of holo-ACP

The classical model of fatty acid and polyketide biosynthesis pictures a semi-static ACP

with a highly mobile 18-20 Å ‘swinging’ PP arm to deliver intermediates as well as starter

and extender units between catalytic domains. However, recent studies with DEBS and the

mammalian fatty acid synthase have provided compelling evidence that ACP has to interact

with catalytic active sites which are separated by distances as long as 80 Å.85 In fact, in a

round of chain extension in which the nascent β-carbon is fully reduced to saturation (e.g.

module 4 in DEBS and Type I FAS), the ACP-bound substrate must be shuttled

successively from AT to KS, then to KR, DH and ER. Finally, the extended polyketide is

ACP monomers ACP dimer


37

either passed to the KS of the following module (e.g. in DEBS) or cleaved from its anchor

ACP by TE (e.g. in mammalian FAS). Thus as many as six partner enzymes have to be

recognized by ACP. Therefore ACP-partner enzymes interactions are of utter importance to

understand the mechanism of polyketide and fatty acid synthesis.

2.1. Protein-protein interactions in Type II FAS and PKS

Type II fatty acid and polyketide synthases consist of several monofunctional enzymes

which are presumed to form a protein complex stabilized by non-covalent forces (Section

1.2.2). In order to form a Type II PKS-derived polyketide such as actinorhodin, a series of

10-15 proteins need be at the same time in the same place of the cell and interact with each

other efficiently. Thus it holds that efficient protein-protein recognition is vital in Type II

polyketide synthases.

The FAS counterpart of Type II polyketide synthases has been studied thoroughly.

Type II fatty acid synthases also consist of a collection of individual enzymes encoded by

separate genes. The E. coli FAS is the model for Type II fatty acid synthases.101 Very

important structural information has been gathered: to date, the NMR structure of ACP102

and the crystal structures of MCAT,104,105 the elongation β-ketoacyl synthases KASI and

KASII,103,106,107 the initiation ketoacylsynthase KASIII,108 as well as those of KR,109 DH,111

and ER110 have been reported.

Despite this wealth of structural information, little is known about protein-protein

interactions between the Type II FAS components. Seminal work by Zhang, Rock and co-

workers was carried out by in silico modelling of the docking of E. coli ACP on one of the

ketosynthases used by E. coli in the synthesis of fatty acids, i.e. KASIII.111 KASIII

catalyzes the formation of acetoacetyl-ACP from acetyl CoA and malonyl-ACP (Scheme

2.1.A). They identified a positively charged pocket in KASIII in close interactions with

negatively charged residues in helix II of ACP. Significantly, R249KASIII was observed to

interact with E41ACP (which is situated within helix II of ACP and corresponds to E47 in

act ACP). They proposed that, this ion pair had a critical role in orientating the ACP-bound

substrate towards the catalytic active site in KASIII.

Biochemical assays were performed by the same group to validate this model. Zhang,

Rock and co-workers harnessed the fact that KASIII can also accept malonyl CoA instead


38

of malonyl-ACP as a substrate (although much less efficiently), (Scheme 2.1.B). The

hypothesis that holo-ACP could be an inhibitor of this second reaction was addressed.111 In

this model, holo-ACP could dock onto KASIII and inhibit KASIII activity towards malonyl

CoA. In other words, if holo-ACP docked onto KASIII, its PP arm would enter the active

site of KASIII, and the ketosynthase would not be able to process malonyl CoA as a

substrate. When the wild type (WT) KASIII was subjected to this assay, strong inhibition

by ACP was observed (20% activity at 150 µM ACP and 5 mM malonyl CoA). A mutant

R249A KASIII showed 80% activity under the same conditions.111 This lack of inhibition

suggested inefficient binding of ACP to KASIII, and therefore supported the role of R249

in KASIII as binding site for ACP.

ACP--S OH

OO

CoA-S

O ACP--S

OO

CoA

CoA-S OH

OO

CoA-S

O CoA-S

OO

CoA

Scheme 2.2 Reaction scheme of E. coli KAS III. A. Condensation of acetyl CoA and malonyl-ACP to form

acetoacetyl-ACP. B. The same reaction is possible using malonyl CoA instead of malonyl-ACP.

Models for the binding of act ACP onto S. coelicolor MCAT and for the docking of

Helicobacter pylori ACP to MCAT have also been done in silico.113,114 The importance of

charged residues along helix II of ACP, as well as hydrophobic forces, were postulated to

determine these protein-protein interactions.113,115 A further model of B. subtilis ACP

bound to ACPS also appointed helix II of ACP as the docking region upon ACPS.116

Similarly, in silico analysis of the structure of ACP bound to ER in E. coli FAS showed

several acidic residues in and close to the ACP helix II (D41, D44 and E47, act ACP

numbering throughout) interacting with basic amino acids in E. coli ER (L201, R204,

L205).117 Experimental evidence for the role of helix II of E. coli ACP in the binding to E.

coli KR was gathered by the group of Rock.118 Using a combination of inhibition studies,

A.

B.

KAS III

KAS III


39

surface plasmon resonance, FRET assays and NMR titrations, a pair of aspartate residues

on helix II of ACP (D37 and D41) and the conserved serine S42 as well as I60 in the loop

between helix II and helix III were identified as the docking residues of ACP.118 The group

of Rock then hypothesized that highly conserved negatively charged residues in helix II are

a universal recognition motif that could be responsible for electrostatic interactions between

ACP and its partner enzymes. In a recent study by Cronan, the importance of negatively

charged and hydrophobic residues in helix II of E. coli FAS ACP has also been shown in

vivo.119 Scarcer information is available in Type I fatty acid and polyketide synthases,

although it has been proposed that, electrostatic (via recognition helix II) and hydrophobic

forces are the key players in protein-protein interactions in DEBS73,92 and yeast FAS.121

In fact, the majority of acyl carrier proteins from Type I and Type II FAS and PKS

contain a pair of negatively charged residues six or seven amino acids apart (Figure 2.1).

Within the Bristol group, Arthur studied the interaction of act ACP with act KSα/KSβ, act

KR and S. coelicolor ACPS by tryptophan fluorescence assays. He measured the

dissociation constants of act ACP and a series of mutants, and identified E47 and E53 as

essential residues that support ACP-KSα/KSβ and ACP-KR interactions. E47 was also

involved in ACP-ACPS binding.120

Figure 2.1 Primary sequence alignment of FAS and PKS ACP (green star, the PP attachment serine).


40

2.2. Purification of acyl carrier proteins

All act acyl carrier proteins are mutant proteins in which the cysteine 17 has been mutated

to serine (see introduction to Chapter 2). The acyl carrier proteins used in this work are the

act mutants C17S ACP (referred to as ‘reference act ACP’), E47A/C17S ACP, E47V/C17S

ACP, E53A/C17S ACP and R72A/C17S ACP as well as the acyl carrier proteins from the

griseusin (gris) and daunorubicin/doxorubicin (dps) polyketide synthases.

All acyl carrier proteins come from dual expression plasmids that harbour the

corresponding acyl carrier protein gene as well as the gene encoding E. coli ACPS.47 All

acyl carrier proteins were over-expressed in E. coli BL21 (DE3) and purified to

homogeneity as described previously in the literature for act ACP52 and in the Experimental

Chapter (Section 8.5.2.1). Most of the acyl carrier proteins were obtained in their holo form

as judged by ESMS analysis (Figure 2.2), except E47V ACP, where the ratio holo:apo was

1:2. Phosphopantetheinylation of this ACP was then performed in vitro with purified S.

coelicolor ACPS and CoA (in a ratio of ACPS:ACP:CoA concentrations of 1:10:100) in the

presence of 10 mM Mg2+.42 Final holo:apo relative abundance was 1:1. Separation of the

apo and holo forms of act E47V ACP was achieved by anion exchange chromatography

(Q-Sepharose) and the ACP was eluted as described for ACP purification.

Figure 2.2

2.3. Reduction of ACP dimers

Dithiothreitol (DTT) 45 has been traditionally used to carry out the reduction of the

intermolecular disulphide bond that can occur between the terminal thiols of the PP arms of

holo-ACP in vitro (see introduction to Chapter 2). Incubation of holo-ACP dimer with DTT

(1 mM, 30 ˚C, overnight) yields the active holo-ACP monomer. Within the Bristol group,

act minimal PKS assays where ACP was treated with DTT, and then DTT was removed by

gel-filtration prior to assay, were previously performed.8,62,96,122 Because this is a tedious

and time-consuming procedure, the Bristol55 and other groups have also used DTT in situ at

concentrations between 1 and 2 mM.56,57,58,98,99

However, thiol reducing agents such as DTT can potentially be incompatible with

thiolesters, as they can act as nucleophiles and decrease thiolester intermediate

concentrations by transthiolesterification. On the other hand, tris(2-carboxyethyl)phosphine


41

(TCEP) 46 has been described as an alternative reductant in the literature,123 and recently

Moore and co-workers have used TCEP as reducing agent in PKS assays.124

HS

OH

OH

SH

HO

O

P

OH

O

OHO

45 46

When act holo-ACP is lyophilized in air, it rapidly forms a disulfide dimer via the linking

of the PP thiols.8 Dimer prepared in this way was then treated with TCEP (1 mM) for 1 h at

30 ˚C to test for the ability of TCEP to reduce the disulfide bonds. Subsequent analysis by

ESMS confirmed that >95% of the holo-ACP was in its monomeric form (Figure 2.3).

Figure 2.3 Mass spectrum of the reference act holo-ACP dimer after incubation with TCEP. Expected

masses, 9441 Da (monomer), 18882 Da (dimer).

We then investigated the effect of DTT and TCEP on acylated ACP by ESMS. Malonyl-

ACP was made using the method described by Hitchman for the self-malonylation of

ACP:55 purified act holo-ACP monomer (200 µM) was incubated with malonyl CoA (1

mM) for 2 h at 30 ˚C. Then, the protein solution was desalted by gel filtration to remove the

excess malonyl CoA. About 90% of the total ACP was converted to malonyl-ACP, as

judged by ESMS analysis.

monomer

dimer


42

The desalted malonyl-ACP (50 µM) was treated with TCEP or DTT (1 mM) for 1 h at 30

˚C, and the ACP species then analysed by ESMS. Incubation of malonyl-ACP (9527 Da)

with DTT mostly led to transthiolesterification of the acyl group and production of holo-

ACP (9441 Da) as observed by ESMS (Figure 2.4.A). We could only detect traces of

malonyl-ACP after 60 min indicating almost complete removal of the malonate.

This contrasted to the use of TCEP as reductant -TCEP did not react with malonyl-ACP

(Figure 2.4.B). Thus, stock solutions of holo-ACP were routinely treated with TCEP to

ensure production of ACP monomers, and PKS assays were performed in the presence of

TCEP.

Figure 2.4 A. Incubation of malonyl-ACP with DTT. B. Incubation of malonyl-ACP with TCEP. Expected

masses: 9441 Da (holo-ACP) and 9527 Da (malonyl-ACP).

2.4. Studies on the self-malonylation activity of act holo-ACP.

Act ACP has the ability to catalyze its own malonylation from malonyl CoA, to form

malonyl-ACP (see Section 1.2.3.5 in Chapter 1). Within the Bristol group, a radioactive

assay was implemented to study the kinetics of self-malonylation of act holo-ACP with 14C

labelled malonyl CoA.55 The assay involved the reaction of 14C-labeled malonyl CoA with

holo-ACP over time. At set time points the reaction was sampled, protein precipitated,

excess radio-label washed away, protein resolubilised and 14C incorporation estimated by

scintillation counting. This enabled the kinetic parameters of kcat = 0.34 min-1 and KM = 219

µM to be measured.55

A. B.


43

This assay required discontinuous, individual measurements and used significant amounts

of pure protein. The assay was quantitative, but the very large number of measurements

required, and the use of large amounts of protein made this method impractical for further

routine use, for instance to study mutant acyl carrier proteins.

A second, mass spectrometric assay to measure the extent of self-malonylation was

developed by the Bristol group.60,120 Following the incubation of malonyl CoA with holo-

ACP, the reaction was quenched at time intervals and the protein fraction analyzed by mass

spectrometry. Although this assay did not involve radioactive materials, it was also

discontinuous and not suitable for the determination of kinetic parameters due to the

qualitative nature of protein mass spectrometry. The aim of this section is to develop an

assay amenable for the kinetic study of the self-malonylation of ACP.

2.4.1. Development of the α-ketoglutarate dehydrogenase assay to measure the rate of

self-malonylation

α-Ketoglutarate (or 2-oxoglutarate) dehydrogenase (KGDH) is an enzyme involved in the

citric acid cycle.125 The KGDH complex is composed of multiple copies of three

component proteins: α-ketoglutarate decarboxylase (E1), lipoate succinyltransferase (E2)

and dihydrolipoamide dehydrogenase (E3).125,126 Each enzyme needs a cofactor for catalytic

activity, namely thiamine pyrophosphate (TPP) 47, lipoic acid (lip) 48 and flavin adenine

dinucleotide (FAD) 49 respectively.

Oxidation of α-ketoglutarate 50 is catalysed by the consecutive action of E1, E2 and

E3. First, 50 is decarboxylated by E1, which uses 47 as a cofactor (Scheme 2.3-A). Second,

the succinyl unit is transferred to E2 with partial reduction of 48 and regeneration of the

active E1 complex (Scheme 2.3-B). The succinyl unit is then transferred to coenzyme A

(CoA-SH) 51 by E2 (Scheme 2.3-C).

Regeneration of E2 activity is catalysed by E3, which uses 49 as a cofactor (Scheme

2.3-D). E3 itself is reactivated by reduction of nicotinamide adenine dinucleotide (NAD+)

to form NADH 52, Scheme 2.3-E. Overall, α-ketoglutarate and CoA-SH are converted to

succinyl CoA using and equivalent of NAD+ - it is the production of NADH (from NAD+)

which is then followed spectrophotometrically.


44

A number of groups have used the α-ketoglutarate dehydrogenase complex as a coupling

enzyme for assaying the generation of CoA-SH. For example, Khandekar et al studied the

kinetics of the β-ketoacyl-acyl carrier protein synthase III (FabH) from Streptococcus

pneumoniae.127 Molnos et al coupled the reaction of the E. coli malonyl CoA: acyl carrier

protein transacylase (MCAT, FabD) to α-ketoglutarate dehydrogenase activity.128 More

recently, a Type III PKS from Pseudomonas fluorescens and MCAT from Helicobacter

pylori have been characterised by the same method.129,130 The respective FAS or PKS

reaction generates coenzyme A, which is converted to succinyl CoA by the KGDH

complex (Scheme 2.4).

O

HO

O

O-O

E1(TPP) TPP

O

HO

O

E1CO2

E2-lip(S-S)TPP

O

HO

O

E1 E1(TPP) S

O

HO

O

lip(S-H)-E2

S

O

HO

O

lip(S-H)-E2CoA-SH S

O

HO

O

CoAE2-lip(SH2)2

E2-lip(SH2)2 E3(S-S)-FAD E2-lip(S-S) E3(SH2)2-FAD

E3(S-S)-FADE3(SH2)2-FAD NAD+ NADH H+

Scheme 2.3 Reaction scheme of the KGDH assay.

A.

B.

C.

D.

E.

50


45

O

OH

O

O

HO

O

HO

O

SCoA

NAD+ NADH

CoA-SH

Acetyl or malonyl CoA

Scheme 2.4 Quantitative KGDH assay for CoA-SH. NADH production is followed spectrophotometrically.

2.4.1.1. Development of the method to study the self-malonylation of act holo-ACP

The rate of NADH production can be monitored either by measuring absorbance at 340 nm

(ε = 6230 M-1 cm-1) or fluorescence (λexcitation = 340 nm, λemission = 465 nm). Though

fluorescence measurements have increased sensitivity, the ready availability of an

ultraviolet absorbance plate reader in our laboratory led us to choose the latter method.

The concentrations of all assay components were as described in the Experimental

Chapter (Section 8.7.4). To ensure the coupling enzyme was in its holo form, KGDH was

incubated with KGA, TPP and NAD+ for 5 minutes before the assays were started. Previous

studies had reported an initial burst of activity when assays were started by addition of

malonyl CoA, presumably due to small amounts of CoA-SH in malonyl CoA stocks.128,130

In order to avoid this, all assays were started by addition of the enzyme, holo-ACP.

2.4.1.2. Kinetics of the self-malonylation of act holo-ACP

We aimed to adapt the KGDH assay to the kinetic study of the self-malonylation of ACP.

First, we incubated all assay components with KGDH and started the experiment with act

holo-ACP in several concentrations. We observed an increase in the absorbance at 340 nm

due to reduction of NAD+. The initial change in absorbance was dependent on the initial

ACP concentration, and all reactions were complete in ten minutes (Figure 2.5).

FAS or PKS component

KGDH


46

The self-malonylation reaction produces 1 mol of malonyl-ACP and CoA-SH per mol of

holo-ACP and malonyl CoA (see Scheme 1.12 in Chapter 1). If the concentration of

malonyl CoA is in excess, then the equilibrium will lie on the right side and the final

concentration of malonyl-ACP will be essentially identical to the initial holo-ACP

concentration. In turn, the KGDH assay produces 1 mol of NADH per mol of CoA-SH

(Scheme 2.3). Thus it holds that the molar concentration of NADH generated must be equal

to the concentration of malonyl-ACP, and the change in absorbance at 340 nm due to

production of NADH can be directly related to the concentration of malonyl-ACP.

Therefore this method provided a very accurate measurement of the final concentration of

malonyl-ACP and also of the initial concentration of holo-ACP.

Figure 2.5. Raw data for the KGDH assay at initial holo-ACP concentrations of 15 µM (squares), 30 µM

(triangles) and 50 µM (circles). Malonyl CoA concentration was fixed at 1mM.

A kinetic study of the self-malonylation of act holo-ACP was carried out by measuring the

initial reaction rate at a range of substrate concentrations. Malonyl CoA concentration was

varied from 10 to 1000 µM at a fixed holo-ACP concentration of 60 µM and kinetic

parameters calculated according to the Michaelis-Menten model. A turnover number kcat =

50 µM holo-ACP

30 µM holo-ACP

15 µM holo-ACP


47

0.49 ± 0.01 min-1 and Michaelis constant KM = 207 ± 29 µM were determined by direct fit

of the experimental data points to a hyperbolic function (Figure 2.6).

Figure 2.6 Determination of Michaelis-Menten kinetic parameters of the self-malonylation of holo-ACP by

the KGDH coupling system. A. Plot of reaction rate vs. substrate concentration and hyperbolic fit. B. Hanes

linear plot of the data.

2.4.2. Actinorhodin minimal PKS to measure self-malonylation rate

Kinetic analysis of Type II minimal PKS have been published by the Bristol and other

groups. In Bristol, an HPLC method to detect SEK4 and SEK4b was used.8 A radioactive

thin layer chromatography method has been used by other groups to measure polyketide

production by a variety of Type II PKS.56,98,99,131 However, these assays are unsuitable for

kinetic assays because they are discontinuous, thus only allow to measure the extent of

reaction at discrete time points.

Instead, we aimed to monitor the production of the octaketides directly and

continuously. We reasoned that formation of aromatic octaketides could be directly

observable using ultraviolet spectrophotometry.

2.4.2.1. Development of the method

All spectrophotometric measurements were carried out in an absorbance plate reader, as

described in Section 8.7.6. SEK4 and SEK4b were isolated from Streptomyces coelicolor

CH999/pSEK4 following published procedures8,24,34 and used as standards for calibration.

A. B.


48

The absorbance spectra of SEK4 and SEK4b show maxima in the aromatic region at 280

nm. However, the adenine group of CoA also absorbs strongly at this wavelength at pH =

7.3 and A280 was not appropriate for polyketide detection in minimal PKS assays. On the

other hand, our assays rely on the absorbance of SEK4 and SEK4b at 293 nm. The

detection limit of polyketides was estimated as 100 pmol (corresponding to a change of 6

mAU under our experimental conditions), and the calibration was linear up to 30 nmol

SEK4/4b (corresponding to 150 µM polyketides in 200 µl experiments, and a change in

absorbance of 1.89 AU).

The extinction coefficients of SEK4 and SEK4b at 293 nm were determined

experimentally as 13,200 and 12,100 M-1 cm-1 (measured in 100 mM phosphate buffer, pH

= 7.3) respectively. The Bristol and Stanford groups have shown by HPLC analysis of the

products of actinorhodin minimal PKS assays that the ratio SEK4:SEK4b is approximately

1:1 in actinorhodin minimal PKS assays in vitro;8,34 thus a mean extinction coefficient of

12,600 M-1 cm-1 was assumed for kinetic measurements.

The KSα/KSβ heterodimer was purified as described in the Experimental section. Typical

yields were 75-100 mg L-1. When we incubated the act minimal PKS with malonyl CoA, an

increase in the absorbance at 293 nm was observed, due to the assembly of the aromatic

SEK4 and SEK4b (Figure 2.7). A change in A293 was only observed in the presence of

malonyl CoA, holo-ACP and KSα/KSβ; and controls in which individual assay

components were absent did not show any activity.

Having established the detection conditions, we first investigated the dependence of

polyketide production rate on the concentration of KSα/KSβ. Thus, the concentration of

KSα/KSβ was varied while maintaining holo-ACP and malonyl CoA concentrations fixed

at 80 µM and 1 mM respectively; and we observed the reaction at 293 nm. The initial

change in absorbance (i.e. the initial reaction rate) depended on the concentration of

KSα/KSβ (Figure 2.7).


49

Figure 2.7. Increase in the absorbance at 293 nm in act minimal PKS assays. Holo-ACP and malonyl CoA

concentrations were fixed at 80 and 1000 µM respectively. KSα/KSβ was 0.3 µM (diamonds), 0.5 µM

(triangles) and 1 µM (circles).

This experiment was repeated for a variety of holo-ACP and KSα/KSβ concentrations, and

initial reaction rates were measured (Figure 2.8). For KSα/KSβ concentrations less than 0.5

µM, initial reaction rates increased linearly with the concentration of the ketosynthase

components. In these conditions, initial rates depended on the concentrations of both

KSα/KSβ and holo-ACP. At higher KSα/KSβ concentrations the reaction rate depended

solely on the concentration of holo-ACP, indicating that a holo-ACP dependent process

(i.e. self-malonylation) was rate-limiting at concentrations of KSα/KSβ higher than 2 µM.

These results indicated that the actinorhodin minimal PKS followed a two-step mechanism.

In this model, first holo-ACP self-malonylated (from malonyl CoA) to form malonyl-ACP.

In a second step, KSα/KSβ catalyzed the synthesis of SEK4 and SEK4b from malonyl-

ACP (Scheme 2.5). Therefore, KSα/KSβ (at concentrations higher than 3 µM) could

effectively be used as a coupling enzyme for the production of malonyl-ACP in order to

study the kinetics of self-malonylation. In these circumstances, production rates of aromatic

polyketides could be related to the self-malonylation rate by a stoichiometric factor of 8.

1 µM KSα/KSβ

0.5 µM KSα/KSβ

0.3 µM KSα/KSβ


50

Figure 2.8 Dependence of initial rates of octaketide production on the concentration of KSα/KSβ at varying

ACP concentrations: squares, 10 µM; crosses, 25 µM; triangles, 50 µM; circles, 80 µM ACP.

ACP-S OH

O O

holo-ACP

KS!/KS"

HO

O

O

OH

O

HO

O

OHO

O

OH

HO

O

O

CoA-S OH

O O

CoASH

19 27

Scheme 2.5 Two-step mechanism for the actinorhodin minimal PKS. 1. Self-malonylation of holo-ACP. 2.

Polyketide synthesis from malonyl-ACP.

2.4.2.2. Kinetics of the self-malonylation of act holo-ACP

In order to determine the kinetics parameters for self-malonylation of act holo-ACP,

KSα/KSβ and ACP concentrations were fixed to 3 and 60 µM, respectively. Reaction rates

80 µM holo-ACP

50 µM holo-ACP

25 µM holo-ACP

10 µM holo-ACP

2.

1.


51

were then studied for a range of malonyl CoA concentrations (Figure 2.9). Kinetic

parameters of kcat = 2.30 ± 0.27 min-1 and KM = 215 ± 66 µM malonyl CoA were measured

by direct fit to a hyperbolic function.

Comparison of self-malonylation rates determined by the KGDH (Figure 2.6) and the

KSα/KSβ (Figure 2.9) coupling systems indicated that the rate of ACP self-malonylation

increased by a factor of five when KSα/KSβ was present in saturating concentrations, due

to a change in the kcat component of the rate equation. As a control, we used the KGDH

assay to measure the rate of self-malonylation of holo-ACP in the presence of a mutant

KSα/KSβ (AQ mutant) which is incapable of producing polyketides. Under these

conditions, the catalytic number was calculated as kcat = 2.4 min-1. Again, this is ca. 5-fold

higher than the rate of self-malonylation in the absence of the ketosynthase components.

Figure 2.9 Determination of Michaelis-Menten kinetic parameters for the self-malonylation of holo-ACP by

the KSα/KSβ coupling system. A. Plot of reaction rate vs. substrate concentration and hyperbolic fit. B. Hanes

linear plot of the data.

2.5. Studies on the mechanism of self-malonylation of ACP

The mechanism of the self-malonylation reaction of holo-ACP has not yet been fully

elucidated. Early studies speculated that conserved arginine residues within the ACP were

able to bind malonate moieties via salt bridges between the guanidine and carboxylate

A. B.


52

functionalities (Figure 2.10).55 This could immobilize the malonyl group and allow the PP

thiol of ACP to intercept it.

O

CoA-S

O

O

HN

H

NH

HN

ACP

Figure 2.10 Interaction between arginine residues and malonate units.55

A primary sequence alignment of FAS and PKS acyl carrier proteins shows a high degree

of similarity (Figure 2.1). Notably, there is a conserved arginine residue in helix IV of PKS

acyl carrier proteins that is not present in FAS acyl carrier proteins (G74 in S. coelicolor

FAS ACP and R71, R73, R72 and R71 in fren, gris, act and otc acyl carrier proteins

respectively).

Within the Bristol group, ESMS was used to estimate the rates of self-malonylation of

act ACP by following the conversion of holo-ACP to malonyl-ACP.120 Although ESMS

cannot be used to determine precise kinetic parameters, it is useful for qualitative

comparisons between different mutant acyl carrier proteins. Using this method, a R72A

mutant ACP was shown to self-malonylate at a lower pace than the reference act ACP.

Using the continuous method which utilizes KGDH as a coupling enzyme to self-

malonylation, we determined the kinetic parameters for the self-malonylation of act R72A

ACP as kcat = 0.22 ± 0.04 min-1 and KM = 302 ± 63 µM; i.e. kcat was reduced by half with

increased KM when compared with act ACP.

We then investigated if the CoA moiety of malonyl CoA was also important in the self-

malonylation reaction. If the CoA moiety was involved then CoA-SH itself should inhibit

self-malonylation. Of the two spectrophotometric methods we have developed to measure

the kinetics of self-malonylation, only the coupling of SEK4/4b production to generation of

malonyl-ACP could be used in this investigation, as the KGDH assay uses CoA-SH as a

substrate. Thus, KSα/KSβ in high concentrations was used to measure the self-

malonylation activity of holo-ACP in the presence of several CoA-SH concentrations. We

observed a decrease in rate when increasing CoA-SH concentrations were added to our


53

assays. CoA-SH showed a mixed inhibition pattern of self-malonylation, and inhibition

constants of Kic = 72 ± 5 µM and Kiu = 650 ± 150 µM were determined (Figure 2.11).

To rule out the possibility of KSα/KSβ being inhibited by CoA-SH, four control assays

were executed in the presence of malonyl CoA: ACP transacylase (MCAT), so that the rate

of KSα/KSβ could be measured (see Chapter 3). No inhibition of KSα/KSβ was

measurable at a CoA-SH concentration of 2 mM.

Figure 2.11 Determination of Kic and Kiu for the inhibition self-malonylation of ACP by coenzyme A (open

triangles, 800 µM; filled triangles, 400 µM; open circles, 200 µM; filled circles, 100 µM).

2.5.1. Mechanism of acceleration of self-malonylation rate by KSα /KSβ.

In general, the tertiary structure of ACP is composed of three major helices (plus a shorter

helix between the second and the fourth helices in some models) arranged around a central

hydrophobic core. In the majority of acyl carrier proteins, helix II contains a highly

conserved pair of negatively charged residues six amino acids apart so placing them on the

same face of the helix (E47 and E53 in act ACP). These residues have been proposed to

have a role in protein-protein interactions (see introduction to Chapter 2).

We studied three mutant act acyl carrier proteins available within the group, i.e. E47A,

E47V and E53A ACP in terms of their self-malonylation ability. The three mutations

introduced a hydrophobic residue instead of a negatively charged glutamate. Previous

studies within the Bristol group had shown that these mutant acyl carrier proteins self-

malonylated essentially as the reference act ACP.60,120 Moreover, E47 and E53 are located

A. B.

Kic Kiu


54

on helix II of act ACP, within the proposed recognition motif of ACP. Therefore these

mutants presented a good chance to study ACP: KSα/KSβ interactions during the self-

malonylation step.

First, we evaluated the rate of self-malonylation of these acyl carrier proteins in

isolation using the KGDH assay. Surprisingly, the self-malonylation activity of E47-

mutants and E53A holo-ACP was reduced by ten and two-fold respectively when compared

to act holo-ACP, using the same concentration of malonyl CoA. KM for E53A remained

unchanged. Due to the low activity of E47A and E47V ACP, it would have been necessary

to use very high ACP concentrations (>300 µM) to determine KM.

The reason for the disagreement between the results presented here and previous

observations using a mass spectrometry assay is not clear at this stage. In order to obtain

direct physical evidence, we also did the mass spectrometry assay to allow for qualitative

comparison between act ACP and an E47A mutant ACP. Each protein (at 50 µM) was

incubated with malonyl CoA (1 mM) and then malonyl-ACP analyzed by ESMS at time

intervals. In agreement with our spectrophotometric assay (Figure 2.5) the reaction

catalyzed by act ACP reached equilibrium in 5-10 minutes, whereas E47A was much less

efficient (Figure 2.13), (see Appendix III for raw ESMS data).

Figure 2.12 Self-malonylation of the reference act ACP (circles) and E47A ACP (open circles) as measured

by ESMS.

Act ACP

Act E47A ACP


55

So it appeared that we could not use E47A and E53A as control acyl carrier proteins in the

KSα/KSβ assay. We then decided to study the self-malonylation activity of other Type II

acyl carrier proteins such as griseusin (gris) ACP and daunorubicin/doxorubicin (dps) acyl

carrier proteins. Gris ACP possesses the corresponding residues to E47, E53 and R72,

while in dps ACP the two glutamates are conserved and H70 substitutes for the arginine

residue. HPLC analysis of the products of act minimal PKS with these acyl carrier proteins,

as well as with E47A, E47V and E53A act acyl carrier proteins, showed that all acyl carrier

proteins were able of sustaining SEK4 and SEK4b production (and thus self-malonylated)

to some extent (Figure 2.14).

Figure 2.13 HPLC analysis of SEK4 and SEK4b produced in 2 h by KSα/KSβ (0.3 µM) and a series of ACP

(50 µM). From bottom to top, the reference act ACP, dps ACP, E53A ACP, R72A ACP, E47A ACP, E47V

ACP, gris ACP. Yields after 2 h were 5.5, 2.3, 2.0, 2.3, 0.6, 0.1 and 0.04 pmol SEK4 and SEK4b,

respectively.

We estimated kcat values simply by measuring self-malonylation rates at various

concentrations of holo-ACP using both the KGDH and the KSα/KSβ assays. At high

substrate concentrations (when compared to KM values), the slope of this plot corresponds

to the catalytic number, kcat. Thus, malonyl CoA concentration was fixed to 1 mM, i.e. 5

times KM. Although this assay underestimates kcat, it is useful to provide a general picture of

SEK4 SEK4b

Gris ACP

E47V ACP

E47A ACP

R72A ACP

E53A ACP

Dps ACP

Act ACP


56

self-malonylation rates, as long as the affinity of the mutant ACP for malonyl CoA is

similar to the reference act ACP. As a control, we studied the self-malonylation of act ACP

by this method. kcat was underestimated by 8% (KGDH assay) and 12% (KSα/KSβ assay).

With this restriction in mind, we determined kcat for WT gris ACP, WT dau ACP and act

mutants E47A, E47V, E53A and R72A ACP (Table 2.1).

ACP Act E47A E47V E53A R72A Gris Dps

kcat (min-1) KGDH 0.45 0.05 0.02 0.27 0.22 0.04 0.23

kcat (min-1) KSα /KSβ 2.03 0.09 <0.06 0.12 0.16 <0.06 0.23

Table 2.1 kcat values for the self-malonylation of ACP as measured by KGDH and the KSα/KSβ assays.

Two conclusions could be drawn from the data in Table 2.1. First, that the self-

malonylation rate did not increase in the presence of KSα/KSβ with either the act mutants

or gris or dps ACP. Second, that the self-malonylation activity of act E53A ACP and dps

ACP in isolation (as measured by the KGDH assay) was around half that of the reference

act ACP, whereas E47A, E47V and gris ACP self-malonylated more slowly, at between 5

and 10% of the rate of act ACP.

2.6. Discussion

2.6.1. Studies on the quality of ACP prior to reaction

In order to undertake a kinetic study of a Type II PKS, it is essential to assess the levels of

holo-ACP vs. apo-ACP, the levels of dimeric and monomeric holo-ACP species present in

act minimal PKS assays and the extent of acylation of the monomeric holo-ACP

component prior to reaction. This may be achieved by ESMS.

Here we have presented evidence that the traditional disulfide reductant DTT is

incompatible with malonyl-ACP as incubation of DTT with malonyl-ACP resulted in

production of holo-ACP due to transthiolesterification (Scheme 2.6). Thus, DTT reduces

the concentration of thiolester intermediates such as malonyl-ACP in polyketide


57

biosynthesis and should be avoided in kinetic studies. On the other hand, TCEP is useful as

an alternative reductant. TCEP efficiently reduces ACP dimers and does not react with

malonyl-ACP. Therefore TCEP can be included in PKS assays, whereas DTT must be

avoided especially if the kinetics of the PKS are going to be assessed.

OH

OH

SHHS

ACP

S

O

HO

O

Scheme 2.6 DTT reduces malonyl-ACP concentration by transthiolesterification

2.6.2. Both malonate and CoA are important for binding onto ACP

The mechanism of self-malonylation has been proposed to rely on electrostatic interactions

between the guanidine group of positively charged arginine residues in the surface of ACP

and the carboxylate functionality of the malonate substrate (Figure 2.10).55 This hypothesis

was tested within the Bristol group by the generation of the act ACP mutants R11A, R34A

and R72A. The self-malonylation activity of these proteins was assessed by ESMS. It was

found that, while the self-malonylation activity of R11A and R34A ACP was unaffected,

R72A ACP self-malonylated at around half the rate of the wild type ACP.60 Herein, we

have validated this qualitative estimation by determining kcat for R72A as 0.22 min-1, in

comparison to 0.49 min-1 for act ACP. KM for R72A ACP was 302 µM, in comparison with

207 µM for the reference act ACP. In short, mutation of R72 decreased the specificity

constant kcat / KM from 2.4 mM-1 min-1 to 0.7 mM-1 min-1.

We also studied the importance of the CoA moiety for malonyl CoA: ACP binding. Within

the Bristol group, the binding of malonyl CoA to ACP was studied by NMR. Anna

Szafranska studied the effect of adding malonyl CoA to apo-ACP, and identified a series of

residues whose chemical shift changed with respect to apo-ACP alone.132 No residues on

helix I or the loop between helix I and II were affected; on the other hand, 13 residues

situated on helix II, the loop between helix II and helix III, helix III itself and the loop

between helix III and helix IV were identified as a putative malonyl CoA binding site


58

(Figure 2.15). These residues included hydrophobic (L43, M46, A50, Y56, V58, S59, V64),

positively charged (R67 and R72) and negatively charged (E47, E53, D62, D63, D69)

amino acids.

Figure 2.14 A. Affected residues upon binding of malonyl CoA onto ACP (yellow). B. All affected residues

are solvent-exposed (blue).132

We showed here that CoA-SH competes with malonyl CoA for binding sites within ACP,

thus inhibiting self-malonylation. This is important as CoA-SH itself is produced in the

self-malonylation reaction. The general mechanism for enzyme inhibition is one in which

the inhibitor can bind both to the free enzyme and also to the enzyme-substrate complex

(Scheme 2.9). This general scheme is known as ‘mixed inhibition mechanism’.94

ACP + malonyl CoA ACP.malonyl CoA malonyl-ACP + CoA-SH

CoA-SH

+

ACP.CoA-SH

CoA-SH

+

CoA-SH.ACP.malonyl CoA

KicKiu

+ malonyl CoA

Scheme 2.7 Mechanism that produces mixed inhibition of self-malonylation by CoA-SH.

I

II

III

IV A. B.


59

Kic and Kiu are known as the competitive and uncompetitive inhibition constants,

respectively. We expected CoA-binding to the free enzyme, ACP, to form a complex

ACP.CoA-SH. Our data reported a Kic = 72 µM, which is lower than the Michaelis constant

for self-malonylation of ACP (ca. 214 µM malonyl CoA). This suggests that, the small,

acidic ACP has three times lower affinity for coenzyme A when a negatively charged unit

such as malonate is bound to its phosphopantetheine arm, even taking into account the salt

bridges between R72 and the malonate unit. Unexpectedly, there was an uncompetitive

component in the inhibition, which probably means that ACP can bind two CoA moieties at

any time. This effect, though measurable, was ten times less important than its competitive

counterpart.

2.6.3. KSα /KSβ accelerates malonylation of ACP

We measured the kinetics of self-malonylation spectrophotometrically by two methods.

The α-ketoglutarate dehydrogenase (KGDH) assay measures the production of CoA-SH

from malonyl CoA catalyzed by holo-ACP. Our kinetic data (kcat = 0.49 min-1, KM = 207

µM) are in good agreement with those previously reported by the Bristol group using a

radioactive discontinuous method (kcat = 0.34 min-1, KM = 219 µM).55 The difference in kcat

could be attributed to the use of DTT in previous studies, which is likely to reduce the

concentration of malonyl ACP (Section 2.3).

The second assay to measure self-malonylation of holo-ACP employed KSα/KSβ as a

coupling enzyme for the production of malonyl-ACP. The minimal PKS produces the

aromatic polyketides SEK4 and SEK4b which can be quantitated by their absorbance at

293 nm. Unexpectedly, we observed a change in kcat when self-malonylation was measured

in the presence of KSα/KSβ (from 0.49 to 2.30 min-1) with unchanged KM leading to a five

times higher specificity constant kcat/KM in the presence of KSα/KSβ. This may be due to

either of two possible reasons, i.e. that KSα/KSβ has some acyltransferase activity, or that

KSα/KSβ activates holo-ACP towards self-malonylation.


60

2.6.3.1. KSα/KSβ as a potential acyl transferase

In type II FAS, MCAT catalyzes the malonylation of FAS ACP. In general, type II PKS

lack a dedicated MCAT. When the gene encoding act KSα was sequenced, a putative

acyltransferase motif GHSLG was found in KSα (italics, the putative AT serine active site).

It was then speculated that KSα might be a bifunctional enzyme, exhibiting malonyl CoA:

ACP transacylase as well as β-ketoacylthiolester synthase activities.20 Interestingly,

mutation of the putative serine active site (S347) to leucine reduced the production of

polyketides in vivo, although it did not completely abrogate it.133 At the same time, Meurer

and Hutchinson showed that a mutation S347A (act KSα numbering) in the tetracenomycin

KSα did not have any effect in polyketide production and it was consequently assumed that

AT activity was not an essential feature of KSα.134 Years later, when the self-malonylation

ability of act, gris and otc ACP was discovered by the Bristol group,55 it became clear that

production of polyketides could be supported in the absence of any acyltransferase activity.

The determination of the crystal structure of act KSα/KSβ by the Stanford group

showed that, although S347 was close to the active site cysteine, its potential involvement

in loading was hampered by its buried position within the hydrophobic core of the

enzyme.31

It was conceivable that the increase in observed rate of malonylation of ACP in the

presence of KSα/KSβ could be accounted for if KSα/KSβ were contaminated with S.

coelicolor MCAT. If KSα/KSβ were contaminated with MCAT, then, under conditions

where malonylation was rate limiting, one would expect to observe a decrease in the

measured KM value towards that for the MCAT catalysed reaction (KM of malonyl CoA as a

substrate for MCAT is estimated as ca 60 µM).46 However, the same KM value for malonyl

CoA was measured in the presence and in the absence of KSα/KSβ. Thus we concluded

that the increase in measured kcat value represented a genuine increase in the catalytic

efficiency of the ACP malonylation reaction when the ACP was in the presence of

KSα/KSβ.


61

2.6.3.2. KSα/KSβ activates holo-ACP towards self-malonylation

The alternative to a KSα-catalyzed malonylation of ACP is a KSα/KSβ-mediated

conformational change in ACP structure, which renders ACP more active towards self-

malonylation. NMR and crystal structures of some FAS and PKS acyl carrier proteins (but

not act ACP)30 have shown that some holo-ACP exists in at least two different

conformational states in solution in ratios from 90:10 to 65:35 major to minor

isomers.36,40,41 For instance, it has been shown that Plasmodium falciparum FAS holo-ACP

exists in two different conformational states in solution in a ratio 65:35. This is due to

conformational changes, in particular, of residues V41, A60 and L61.41 These changes have

two important effects on the conformation of ACP: first, whereas in the major

conformation there are three residues that interact with the phosphopantetheine arm (S37 –

the attachment point-, L38 and D39), V41 poses an additional restraint to the movement of

the PP arm only in the minor isomer. Second, helix III (D57-A60) unwinds due to the

changes in A60 and L61, rendering a more flexible loop (Figure 2.16).

Figure 2.15 Ribbon representation of the major (A. PDB file 2FQ0) and minor (B. PDB file 2FQ2)

conformers of P. falciparum FAS ACP. Residues D57 to A60 are highlighted in magenta.

It has been suggested that these two ACP conformations correspond to ‘open’ and ‘closed’

forms of ACP.41,135 In the closed conformation (the major P. falciparum isomer), the loop

A. B.


62

between helices II and III is packed against the three helix bundle and prevents the insertion

of the hydrophobic acyl group into the protein core. In this state, the PP arm is thought to

be flexible and in an extended conformation. In the open conformation, a more flexible

secondary structure due to the unwinding of helix III moves away from the three-helix

bundle, and allows the insertion of acyl chains into the hydrophobic pocket. This is

important because FAS ACP has to shuttle the growing fatty acid chain to the active sites

of enzymes (KR, DH, ER). Consequently, it was assumed that ACP must undergo this

conformational change in each successive cycle of chain elongation.41

The first structure of a PKS ACP was obtained by the Bristol group in 1997. The

actinorhodin apo-ACP was structurally similar to FAS ACP, and was composed of three

major helices (plus a shorter one, comprising residues D62-R67) packed into a bundle, and

a flexible loop between helices I and II. It was reported that act ACP only existed in one

conformation, and it was speculated that conformational variability may only be a feature

of FAS ACP.30 However, the solution structure of frenolicin (fren) PKS ACP also

evidenced two conformational states.39 In this case A56, T58, D59 and G63 were found to

exchange slowly (50-100 ms) between two states. This rendered the loop between helices II

and III as well as helix III itself intrinsic flexibility to determine the conformation of PKS

ACP.

The general fold motif of FAS and PKS acyl carrier proteins is also conserved in a third

group of proteins, the peptidyl carrier proteins (PCP) from non-ribosomal peptide synthases

(NRPS). Peptidyl carrier proteins are also small proteins of ca. 8-10 KDa and, like acyl

carrier proteins, they house a universal serine nucleophilic residue upon which a PP arm is

attached. The role of the carrier protein in polyketide and non-ribosomal peptide

biosynthesis is to shuttle substrates and intermediates between catalytic domains. Whereas

in PKS the substrates are carboxylic acids such as malonate, in NRPS the substrates are

amino acids, for example alanine. In both carrier proteins, substrates are activated as

thiolesters (Figure 2.17).


63

S OH

O OACP

SNH2

OPCP

Figure 2.16 ACP and PCP activate their substrates (for instance, malonate and alanine) as thiolesters.

The tyrocidine 53 NRPS is an assembly line very much like typical modular PKS, in which

several catalytic domains are enclosed in each module, and every module carries out the

elongation of the non-ribosomal peptide via the generation of a new peptide bond between

the lengthening oligopeptide and an extender peptidyl unit. Weber, Marahiel, Holak and co-

workers studied the PCP from module 3 of the tyrocidine NRPS (TyrC3-PCP). Initially, the

structure was released as a four-helix bundle analogous to FAS and PKS ACP.136

Upon recent examination, however, the NMR spectrum of apo-PCP indicated the existence

of two different and slowly exchanging conformations. The changing residues were at the

N terminus of helix II and adjacent loop, including the active site residue S45; and between

helices II and IV. When the holo form was studied, it was found to exist in two

conformations as well. Interestingly, while the apo and the holo forms shared one

conformational state (termed A/H), the two remaining states (A for apo and H for holo)

were distinct from each other and from the structurally identical common state.137

The three states of TyrC3-PCP differ mainly in the orientation and length of the helices.

The purely apo (A) state was achieved when the active site serine was mutated to alanine.

While helices I and II are shortened with respect to the A/H conformation, helix III does

not exist at all. In the A/H state, the helices are more stable and longer, and the loop

between helices II and IV now forms helix III. This is the most compact structure. On the

other hand, in the H state helix III unravels itself and prompts a reorganization of the

globular protein core (Figure 2.18).


64

O

N

O

NH

CH3

H3C

HN

O

HN

NH2O

HN

O

CH3H3C

HN

OH

O

NH

O

NH2

O

NH

HN

O

O

O

NH2

53

Figure 2.17 Ribbon structures of TyrC3-PCP in its A state (A. PDB file, 2GDY), A/H state (B. PDB file

2GDW) and H state (C. PDB file 2GDX). Helices are numbered in each structure.

Biological confirmation of the importance of these different conformers in TyrC3-PCP was

gathered by experiments with the phosphopantetheinyl transferase and thiolesterase partner

enzymes. The former interacts with only apo-ACP naturally, and in fact it could only

recognize apo-ACP in its A state. Complementary, NMR titrations with the thiolesterase

revealed protein-protein interactions between TE and holo-PCP only in its H state.137

I

I

I

II

II

III

II

IV

IV

IV

A. B.

C.


65

Altogether, this information points towards the conformational flexibility of ACP and PCP

as an important mode of controlling the interactions between the carrier protein and partner

enzymes in complex biosynthetic pathways,115 and it could account for our observations

that catalytic properties of ACP are enhanced by the presence of its known counterpart,

KSα/KSβ. In any event, our kinetic studies provide evidence for a close association of ACP

and KSα/KSβ during the self-malonylation step.

2.6.3.3. Insights into ACP-KSα/KSβ interactions leading to enhanced self-

malonylation activity.

In order to gain understanding of the association between ACP and KSα/KSβ during the

self-malonylation step, we proceeded to study act ACP mutants such as E47A, E47V and

E53A in terms of their self-malonylation activity in the presence and absence of KSα/KSβ.

Unexpectedly, neither of the act mutant acyl carrier proteins studied performed as well

as the reference act ACP in isolation, when the self-malonylation was studied by the

KGDH assay. kcat values decreased two-fold (E53A) and 10-fold (E47A and E47V ACP).

This was striking because previous evidence within the Bristol group using an ESMS

assays had shown that act E47A and E53A ACP had similar activities to the reference act

ACP. When we repeated the ESMS assay with our E47A ACP, we observed a clear

reduction in self-malonylation activity, in agreement with our spectrophotometric assay.

It is not clear why E47A and E53A acyl carrier proteins performed as well as the

reference act ACP in the past. In our hands, these acyl carrier proteins were clearly

deficient in self-malonylation. We have shown that the CoA moiety is important for

appropriate ACP- malonyl CoA interactions (Section 2.5), so the possibility that E47 and

E53 are involved in CoA binding, as proposed by Szafranska,132 might be valid. Although it

seems unlikely that negatively charged residues are directly involved in binding the CoA

moiety, they may play an important role in creating a net of hydrogen bonds leading to

efficient interaction between ACP and CoA.

We then included two acyl carrier proteins from other type II PKS, i.e. gris ACP and dps

ACP, in our studies. Gris ACP contains the corresponding residues to E47, E53 and R72

(act ACP numbering). Dps ACP possesses the corresponding E47 and E53 but a histidine


66

residue substitutes for R72. Gris ACP and dps ACP in isolation self-malonylated more

slowly than act ACP. We did not observe any increase in rate in the presence of KSα/KSβ.

In conclusion, E47, E53 and R72 appear to be essential residues to support self-

malonylation of act ACP, both in the absence and in the presence of KSα/KSβ.

Unfortunately, we could not confirm that E47 and E53 are involved in protein-protein

interactions between act ACP and KSα/KSβ because the self-malonylation activity of

E47A, E47V and E53A was impaired when studied in isolation. The study of the self-

malonylation of gris and dps ACP, which possess the equivalent to E47 and E53, suggested

that there are still more features that prompt an efficient ACP: KSα/KSβ association which

have not been identified.

Future work should adapt the KGDH assay for the measurement of NADH production by

fluorescence spectrophotometry. When excited at 340 nm, NADH emits energy at 465 nm

that can be monitored continuously in a fluorescence spectrophotometer. Fluorescence

measurements are intrinsically more sensitive than absorbance measurements, and therefore

it should be possible to use the KGDH assay for the study of the self-malonylation of ACP

mutant proteins by detection of NADH by fluorescence. This is most important for a full

kinetic characterization of acyl carrier proteins with low activity such as act E47A and

E47V ACP, and gris ACP.

The characterization of the activation of self-malonylation by KSα/KSβ should be

continued, first, by the study of more ACP mutants which self-malonylate as the reference

act ACP in isolation. Eventually, residues responsible for ACP: KSα/KSβ interactions in

KSα/KSβ could also be identified.


67

3. Initiation of polyketide synthesis.

Type II FAS systems found in most bacteria consist of a series of discrete proteins, each of

which catalyzes an individual reaction of the fatty acid biosynthetic pathway. In organisms

using a type II pathway, for instance E. coli and S. coelicolor, there are at least two β-

ketoacyl ACP synthase (KAS) enzymes, known as KASII and KASIII.138,139 KASII is the

generic enzyme responsible for the elongations required for synthesis of long-chain fatty

acids,140 analogous to act KSα. KASIII catalyzes the first condensation from acetyl CoA

and malonyl-ACP to produce butyryl-ACP 54 (Scheme 3.1), the starter unit for FAS.141

Butyryl-ACP is then the substrate for the KASII-catalyzed elongations that result in long-

chain fatty acids.142

S

OH

O

O

ACP

S

O

CoA

S

O

O

ACPCO2, CoA-SH

+

54

Scheme 3.1 Initiation of fatty acid synthesis in E. coli, catalyzed by KASIII.142

We have seen in previous chapters that the actinorhodin PKS does not require KASIII

activity, and the only ketosynthase capable of catalyzing carbon-carbon bond forming

reactions is KSα. However, an analogous mechanism to bacterial FAS, in which additional

KASIII (also known as priming ketosynthase), and also AT and sometimes ACP proteins

are responsible for the biosynthesis and attachment of short-chain fatty acids, has been

proposed for other Type II PKS. Particularly, this mechanism has been proposed to operate

in polyketide synthases that employ a starter unit other than acetate.

For example, the daunorubicin (dps) PKS employs a propionyl starter unit (see Figure 1.3

in Chapter 1). How the dps PKS chooses propionate is currently the subject of intense

research.142 Initial in vivo complementation studies were carried out by Meurer and

Hutchinson. They used the KSα and KSβ components from the daunorubicin PKS with

KASIII


68

ACP and tailoring enzymes from the tetracenomycin PKS.144 Only acetyl-derived

polyketides were produced such as 55 (starter unit shown in red), indicating that dps KSα

and KSβ did not have any requirement for propionyl-ACP as a starter unit.

HO

OH OH OH

O

O

OH

55

The same group used cell-free extracts of cultures expressing dps KSα/KSβ, ACP as well

as KR and CYC to feed labelled propionyl-CoA and analyze the metabolites produced.

Incorporation of radioactivity was not observed. When a cell-free extract of a culture

expressing DpsC and DpsD was added, the propionate-derived polyketide UWM5 56

(starter unit shown in red) was produced. This result suggested that DpsC and DpsD were

essential for substrate specificity in cell-free systems.145 Hutchinson are co-workers then

hypothesized that DpsC and DpsD (homologues to bacterial KASIII -although substituting

the active site cysteine for serine- and AT, respectively), were the primary determinants of

starter unit specificity.

O

OH

O

O OH

OH

HO

56

In vitro studies with purified DpsC showed that DpsC could use propionyl CoA as substrate

and was acylated by propionate at Ser118 (Scheme 3.2.A).146 DpsC also catalyzed the

condensation of propionyl CoA and malonyl-ACP to form β-ketovaleryl-ACP 57 (Scheme

3.2.B).146 Subsequently, the valerate unit would be loaded onto KSα for further elongation

with malonate units (Scheme 3.2.C).


69

S

O

CoA

HS

DpsC

S

O

DpsC

S

OH

O

O

ACP

S

O

DpsC

S

O

O

ACP

S

O

O

ACP

S

O

KS!

O

S

OH

O

O

ACP

OMe

O

O

OH

OH

O

O

OH

O

NH2HO

HS

KS!

Scheme 3.2 Proposed mechanism for starter unit specificity in dps PKS. A. Loading of DpsC with propionate.

B. Initiation of polyketide synthesis. C. Elongation of the polyketide chain.147

Other examples of related PKS priming mechanisms include the biosynthesis of frenolicin

(frn), benastatin 58 (ben) and R1128.148,149,150 In all cases, genes encoding KASIII

homologues were identified in the respective gene clusters (FrnI, BenQ and ZhuH

respectively).

In the case of the R1128 PKS, an additional enzyme, i.e. ZhuC (which corresponds to

DpsD in the daunorubicin PKS) was initially appointed a malonyl CoA: ACP transacylase

activity.151 Later, however, the role of ZhuC was revised: when it was added to tcm

minimal PKS assays in vitro, hexanoyl-ACP was preferred to acetyl-ACP as the starter

unit, unequivocally pointing towards a role for ZhuC in initiation of polyketide synthesis.

Tang, Khosla and co-workers proposed that ZhuC (and by analogy DpsD) are thiolesterases

that target acetyl-ACP selectively thus eliminating competitive acetate substrates. Acetyl

and propionyl-ACP were indeed 5, 100 and 200 times better substrates for ZhuC than

butyryl, hexanoyl and malonyl-ACP, respectively.152

Furthermore, an additional ACP (ZhuG, termed priming ACP) has been shown to be

essential for the incorporation of non-acetate starter units in the R1128 PKS. This does not

A.

B.

C.

57

13


70

result in the other ACP being redundant, as the priming ketosynthase ZhuH and the

elongating ketosynthase ZhuA have orthogonal ACP specificity showing preference for the

priming and elongating ACP partners, respectively.98 This is striking because, whereas

there are two acyl carrier proteins in the frenolicin PKS gene cluster, only one ACP is

present in dps and ben PKS. OHO

HO

OHOOH

HO

58

Neither the frenolicin, benastatin and R1128 gene clusters encode the necessary KR, DH

and ER catalysts for the reduction of the β-ketoester generated by KASIII to the butyrate

(frn), hexanoate (ben) or (iso)butyrate, valerate or 4-methylvalerate (R1128 complexes)

starter units (see Figure 1.3 in Chapter 1), which has led to the assumption that these

activities are recruited from fatty acid metabolism.149,151 In remarkable work, Khosla and

co-workers harnessed the fact that, when the R1128 gene cluster is heterogously expressed

in S. coelicolor, it produces the same polyketides as in the original producer (S. sp. R1128),

to identify and purify an enzyme (SCO1815) that reduces the ACP-tethered β-ketoacyl

substrates produced by the initiation module of R1128 PKS. SCO 1815 was highly

homologous to FAS KR.153

A second, alternative priming pathway in Type II PKS that do not use KASIII-like activity,

is the direct loading of the starter unit onto the minimal PKS. For instance, in the enterocin

(enc) PKS from Streptomyces maritimus, it has been proposed that an enzyme with ACP

ligase activity, namely EncN, is capable of loading ACP with the benzoate starter unit

directly from benzoic acid 58 to form benzoyl-ACP 59. The benzoate is then transferred to

KSα.124

OHACP+

SH

O

S

O

ACPS

O

KS!

Scheme 3.3 Priming of the enc PKS with the benzoate starter unit.124

EncN KSα/KSβ 58 59


71

The priming of Type II PKS with acetate units proceeds in a different manner, also

different to fatty acid biosynthesis. First, ACP is loaded with malonate from the

corresponding CoA thiolester either by self-malonylation or via MCAT (see Sections

1.2.3.2 and 1.2.3.5). Malonyl-ACP is then decarboxylated to acetyl-ACP and transferred to

KSα to yield acetyl-S-KSα and initiate polyketide building. This mechanism is unique in

fatty acid and polyketide synthases in that it generates acetyl-ACP. This differs from FAS

where the first ACP-tethered intermediate is acetoacetyl-ACP (see Scheme 1.1 in Chapter

1).

The mechanism for decarboxylation of malonyl-ACP to generate acetyl-ACP has been a

matter of debate in the literature. This originated from the discovery of the ‘mystery’ gene

encoding KSβ,20 which was then thought to occur only in Type II PKS systems (later,

similar KSQ proteins were described in some modular Type I PKS). Initial in vivo

experiments by Hopwood showed that a mutation in KSβ abolished polyketide production

in actinorhodin-producing S. coelicolor.154 Once established that functional KSβ were

essential for PKS activity, the Stanford group speculated that KSβ provided the template

for the growing polyketide chain and was the principal determinant of chain length (see

Section 1.2.3.3 in Chapter 1).

Thus, in the Stanford model for the act minimal PKS (see Section 1.2.3.4 in Chapter 1)

KSβ exerts a structural (rather than catalytic) role. Based on the crystal structure of the

KSα/KSβ complex, the Stanford group proposed that the corresponding residue to the

cysteine active site of KSα in KSβ (Q161) was buried and had a structural role, i.e. the

formation of a salt bridge between D297 and R332 (KSβ residues). By default, they argued

that decarboxylation of malonyl-ACP must be catalyzed by KSα.31 This would proceed

with the aid of two histidines (H309 and H346, KSα residues), in analogy to the

mechanism proposed for the yeast FAS,155 KASII from E. coli156 and similar to that in the

chalcone synthase.157

However, biochemical studies with a series of mutant KSα/KSβ within the Bristol and

Cambridge groups in vitro suggested that Q161 in KSβ was essential for efficient malonyl-

ACP decarboxylation activity of the ketosynthase complex. KSα/KSβ mutant complexes

were generated in which either active site (C169 in KSα or Q161 in KSβ) were replaced


72

with an alanine residue.62,122 The following notation of mutant proteins was devised: each

protein complex was termed according to the corresponding residue in the active site of

KSα and KSβ. For example, the wild type KSα/KSβ was referred to as CQ, as there is a

cysteine in the active site of KSα and a glutamine in the active site of KSβ. The following

mutant proteins were generated: AQ, CA and AA KSα/KSβ in which one or the two active

sites of KSα and/or KSβ were replaced by an alanine residue.

First, these mutants, as well as the WT complex, were tested for their ability to produce

polyketides. Thus, each mutant was incubated in vitro with malonyl CoA and holo-ACP

and the products analyzed by HPLC. As expected, only the CA KSα/KSβ mutant was able

to produce polyketides; however, there was a lag of about 5 minutes without activity. This

lag was not observed in the control experiment using the wild type CQ KSα/KSβ. When

external acetyl-ACP was added to the assay using CA KSα/KSβ, the lag was

abolished.62,122 This observation leads to important conclusions. First, that KSα can be

primed with acetate directly by adding acetyl-ACP into solution. This may suggest that the

acetyl-ACP generated by decarboxylation of malonyl-ACP is probably released into

solution, rather than transferred directly to the KSα active site (Scheme 3.4). Second, that

the concentration of acetyl-ACP appears to build up to a certain value before polyketide

synthesis could occur, which suggests that the decarboxylation activity of the CA KSα/KSβ

is not high enough to provide this critical acetyl-ACP concentration at the beginning of the

assay.

S

O

ACP

KS! KS"

S

OH

O

O

ACPS

O

ACP

Scheme 3.4 Two possible models for transfer of acetyl-ACP from the KSβ to KSα active sites.

The fact that acetyl-ACP was released into solution after decarboxylation of malonyl-ACP

was harnessed by the Cambridge and Bristol groups to estimate the malonyl decarboxylase

Bulk solution

Acetyl-ACP released into bulk solution

‘Internal’ transfer of acetyl-ACP


73

activity of the AQ and AA KSα/KSβ, which are incapable of polyketide synthesis.62,122

These mutants were incubated with malonyl-ACP, and the relative abundance of acetyl-

ACP after a certain period of time was determined by mass spectrometry by calculating the

ratio malonyl-ACP: acetyl-ACP. Whereas AQ KSα/KSβ could decarboxylate malonyl-

ACP at 20 µM h-1, the AA mutant did not show any activity towards malonyl-ACP. This

latest result, however, was not reproducible in the Cambridge group: an AA mutant was

later shown to decarboxylate malonyl-ACP essentially at the same rate as the AQ

complex.158

Alternative starter units, such as butyryl-ACP and hexanoyl-ACP, have also been fed to

the act minimal PKS. These acyl-acyl carrier proteins were utilized as substrate by KSα

only when the CA KSα/KSβ was used. When the WT (i.e. CQ) KSα/KSβ was used, the

‘fake’ starter units were out of compete and only acetate-derived polyketides were

detected.96

Taken together, these results showed that KSβ (via its active site Q161, and maybe

D297 and R332, the corresponding residues to the KSα catalytic triad Cys169-His309-His346)

initiates the synthesis of polyketides catalysing the first decarboxylation of malonyl-ACP to

generate the starter unit, acetyl-ACP. This mechanism was also proposed to operate in the

loading module (KSQ) of Type I modular PKS, such as those of monensin 19, tylosin 18

and niddamycin 17.62,122 Once KSα has been primed with an acetyl unit, it catalyses the

condensation of seven further acetates derived from malonyl-ACP.

The ultimate evidence towards the elucidation of the role of KSβ should rely on the

purification of this protein on its own, and subsequent structural studies and biochemical

assays in vitro. However, KSα and KSβ form intimate contacts with each other (more than

20% of their respective surface areas are buried)31 and attempts to purify KSα and KSβ

individually have been unsuccessful.142

The hypothesis, introduced by the Bristol and Cambridge groups, that KSβ is responsible

for the initiation step, was taking here as the starting point. The aim of Chapter 3 is to adapt

the continuous method we developed in Section 2.4.2 to measure the production of

polyketides by the act minimal PKS, to the kinetic study of the reactions catalyzed by KSα

and KSβ.


74

3.1. Kinetics of KSβ can be studied in the presence of MCAT

In Section 2.4.2 we developed a continuous, spectrophotometric method to study the

kinetics of self-malonylation of ACP by coupling the formation of malonyl ACP to the

production of SEK4 and SEK4b by KSα/KSβ. However, this experiment did not provide

useful information about the kinetics of KSα/KSβ itself. In order to be able to measure the

rate of the reaction catalyzed by KSα/KSβ, we needed to find the appropriate conditions in

which the rate limiting step in the overall reaction executed by the act minimal PKS was

not self-malonylation of ACP, but turnover of malonyl-ACP by the ketosynthase complex.

3.1.1. MCAT accelerates the rate of malonylation of holo-ACP

The Bristol group reported that MCAT accelerates the transfer of a malonate group from

malonyl CoA to the PP arm of act holo-ACP, when compared to self-malonylation at low

concentrations of ACP (see Sections 1.2.3.2 and 1.2.3.5 in Chapter 1).46 Matharu studied

the production of polyketides by the act minimal PKS and found that self-malonylation was

the likely rate limiting step in the overall reaction; and that the presence of MCAT (5 nM)

increased the rate of polyketide production only when ACP concentration was lower than

20 µM.8 In the light of this information, we aimed to compare SEK4/4b production by the

act minimal PKS, in the presence and absence of MCAT. MCAT was thus expressed in E.

coli and purified following literature procedures,46 and characterized by ESMS (Figure 3.1).

Figure 3.1 Mass spectrum of MCAT (expected, 34113 Da); also detected as the potassium adduct.


75

As described previously (Section 2.4.2), our assays rely on the direct spectrophotometric

observation of the polyketides produced by the act minimal PKS. The rate of polyketide

synthesis was studied by determining initial changes in the absorbance. We thus generated

reaction rate data for several ratios of ACP: KSα/KSβ concentrations (see Figure 2.8 in

Chapter 2).

In order to compare polyketide production rate in the presence and absence of MCAT, a

concentration of 0.3 µM KSα/KSβ was chosen which lay within the linear range of the

dependence of the rate with enzyme concentration (see Figure 2.8 in Chapter 2). This meant

that the polyketide synthesis step was rate limiting. Initial rates of reaction were then

measured for a range of holo-ACP initial concentrations. In the absence of MCAT, a linear

dependence of the rate on the concentration of holo-ACP was observed for ACP

concentrations lower than 50 µM (Figure 3.2). This indicated that, under these conditions,

an ACP dependent process was the rate limiting step, which agreed with the hypothesis that

self-malonylation of ACP was the slowest step in the actinorhodin minimal PKS in vitro.

In order to measure the kinetic parameters for malonyl-ACP as a substrate for KSα/KSβ,

the rate of ACP malonylation was increased so that this was no longer the rate limiting step.

This was achieved by using a high concentration of MCAT. We therefore conducted assays

at 0.3 µM KSα/KSβ with various concentrations of holo-ACP, in the presence of MCAT

(0.5 µM). Under these reaction conditions, the concentration of malonyl-ACP in the assays

was constant and equal to the initial, known concentration of holo-ACP. The same

maximum velocity Vmax was eventually achieved both in the absence and presence of

MCAT (Figure 3.2), as the catalysis by KSα/KSβ was not affected by the malonylation of

ACP.

However, at low ACP concentrations the formation of octaketides was much faster in

the presence of MCAT. This confirmed earlier observations by the Bristol group using an

HPLC assay.8 Kinetic parameters for the reaction catalyzed by KSα/KSβ were determined

as kcat = 20.6 ± 0.9 min-1 and KM = 2.39 ± 0.38 µM malonyl-ACP by direct fit to a

hyperbolic Michaelis-Menten equation (Figure 3.2).

To independently determine the turnover number (kcat) of the ketosynthase, we measured

the dependence of the reaction rate on the concentration of KSα/KSβ when MCAT (0.5


76

µM) was added to the assays. At saturating ACP and MCAT concentrations, initial rates

were linear with increasing concentration of KSα/KSβ even at the highest concentration (3

µM), (Figure 3.3). From the slope of this plot, the catalytic constant kcat was independently

calculated as 16.9 ± 0.9 min-1.

In this section we have shown that the kinetics of polyketide production by KSα/KSβ can

be studied in MCAT-supplemented act minimal PKS. Nevertheless, in this assay we were

unable to de-couple the reactions catalysed by KSβ (decarboxylation of malonyl-ACP) and

KSα (elongation of the chain), so we decided to study this further.

Figure 3.2 A. Plot of reaction rate vs. holo-ACP concentration for act PKS ‘minimal’ assays (open circles)

and MCAT-supplemented assays (filled circles). B. Hanes linear plot of the MCAT-supplemented rate data.

Figure 3.3 Upon addition of MCAT, reaction rates become linearly dependent on KSα/KSβ concentrations.

A. B.


77

3.1.2. Initiation of polyketide synthesis is slower than chain elongation

Initiation of polyketide synthesis proceeds via decarboxylation of malonyl-ACP to acetyl-

ACP. If this were the rate-limiting step in the production of SEK4 and SEK4b from

malonyl-ACP, then addition of exogenous acetyl-ACP to MCAT-supplemented act

minimal PKS assays would increase the overall rate of production of polyketides. In other

words, if the concentration of acetyl-ACP were limiting, then addition of external acetyl-

ACP would enhance the overall reaction rate.

To test for this hypothesis, acetyl-ACP was made by the method of Bisang simply by

incubating TCEP-treated holo-ACP (500 µM) with 1-acetylimidazole (5 mM).62 This

reaction was monitored by ESMS. Acetylation of holo-ACP was complete after 2 h (Figure

3.4).

Figure 3.4 ESMS of acetyl-ACP (expected 9483 Da), also detected as the potassium adduct (expected 9522

Da).

Acetyl-ACP was then purified by gel filtration to remove excess 1-acetylimidazole, and

added to PKS assays in increasing concentrations (up to 40 µM). Initial reaction rates were

then measured. In the absence of extra acetyl-ACP octaketides were produced at 4 µM min-

1. When acetyl-ACP was added, polyketide production rates increased (Figure 3.5). This

indicated that acetyl-ACP concentration was limiting under these conditions. When acetyl-

ACP was added in concentrations higher than 20 µM, polyketides were produced ca. 3


78

times faster, and further increase in acetyl-ACP concentration did not produce further

changes in rates (Figure 3.5).

Therefore it seemed likely that in the absence of additional acetyl-ACP the rate limiting

step of the act minimal PKS was the initial decarboxylation of malonyl-ACP catalyzed by

KSβ to form acetyl-ACP. When the PKS was saturated with acetyl-ACP, the new rate

limiting step could possibly correspond to either chain elongation by KSα or the cyclization

and release steps (see Scheme 1.18 in Chapter 1).

Figure 3.5 Change in reaction rate upon titration of acetyl-ACP in MCAT-supplemented act minimal PKS.

KSα/KSβ and malonyl-ACP concentration were 0.25 µM and 20 µM respectively.

3.2. Interaction of ACP and KSβ

The current understanding of protein-protein interactions between the components of

polyketide synthases, especially ACP- partner enzyme association, was reviewed in the

introduction to Chapter 2. In particular, the importance of negatively charged residues in

helix II of ACP was described.

We decided to use the kinetic method developed here to study the kinetics of KSβ to

address this issue. Kinetic parameters for the decarboxylation of malonyl-ACP by KSβ


79

were estimated for a range of act mutant acyl carrier proteins and other Type II acyl carrier

proteins. Mutant acyl carrier proteins such as act E47A, E53A and R72A ACP gave the

same turnover number kcat as the reference act ACP, although the Michaelis constant for

E47A and E53A (and E47V) was 3 times higher (Table 3.1). E47V showed a decreased

catalytic constant. Interestingly, the three WT acyl carrier proteins studied gave the same

specificity constant, kcat / KM, suggesting that act KSβ does not show any specificity for act

ACP in relation to gris and dps ACP.

ACP kcat (min-1) KM (µM) kcat / KM (min-1 µM-1) Act 20.6±0.9 2.39±0.38 8.62±2.37

E47A 20.0±1.9 8.03±2.00 2.49±0.95 E47V 3.5±0.4 6.81±1.05 0.51±0.38 E53A 17.5±1.4 8.07±1.39 2.17±1.00 R72A 20.3±0.8 1.95±0.32 10.41±2.5 Gris ACP 9.5±0.3 1.25±0.17 7.62±1.76 Dps ACP 16.4±1.3 1.86±0.49 8.82±2.65

Table 3.1 Kinetic parameters for the decarboxylation of malonyl-ACP by KSβ (see Appendix 1 for

Michaelis-Menten analysis).

3.3. Discussion

Chain initiation in the act PKS is known to proceed via decarboxylation of malonyl-ACP to

acetyl-ACP. This reaction was previously shown to be catalyzed by KSβ.62 In Chapter 3,

we have used a continuous method to measure the kinetics of this initiation step. This

methodology has been applied to study the interaction of ACP with KSβ.

In the actinorhodin minimal PKS, self-malonylation of ACP is the rate limiting step (See

Chapter 2). Thus, in order to measure the kinetics of the other components of the act

minimal PKS this loading reaction must be accelerated. To this end, we used an enzyme

from the primary metabolism of S. coelicolor, i.e. MCAT, known to malonylate S.

coelicolor FAS and PKS ACP.46 We showed that, upon addition of MCAT (0.5 µM),

loading of the PKS with malonate was no longer rate limiting, and the kinetics of the

ketosynthase complex could be characterized. Furthermore, we were able to de-couple the


80

reactions of KSα and KSβ: we added exogenous acetyl-ACP to MCAT-supplemented

minimal PKS assays and observed a clear increase in rate, meaning that acetyl-ACP

concentration was limiting under these conditions. Therefore it appeared that

decarboxylation of malonyl-ACP catalyzed by KSβ was a slower process than the chain

elongation catalyzed by KSα, and cyclisation and release steps. Thus the kinetic values

determined here (kcat = 20.6 ± 0.9 min-1 and KM = 2.39 ± 0.38 µM) most likely correspond to

the initiation of polyketide synthesis by KSβ.

Previous attempts have been made to measure the kinetics of Type II minimal PKS. For

example, the group of Khosla studied heterologous PKS complexes consisting of the

actinorhodin, tetracenomycin, S. halstedii spore pigment, and pradimycin KSα/KSβ paired

with the frenolicin, R1128, granaticin, S. coelicolor spore pigment and pradimycin

ACP.98,99 In all these cases their assays have relied on the addition of MCAT and are thus

comparable with our assays described above. However, the kcat values reported in their

studies ranged between 0.11 and 0.95 min-1, i.e. at least 20 times lower than those reported

here. Although the reason for this difference is not clear, the fact that they used high

concentrations of DTT in these assays likely reduced the concentration of acyl-ACP species

(see Section 2.3 in Chapter 2). Also, only discontinuous assays (e.g. HPLC, TLC) were

used, and this may have hampered the gathering of genuine initial rates -therefore their

numbers are likely to be underestimations. As expected, there is no difference in the

reported KM values (ranging 1.4-6.4 µM malonyl-ACP), as the determination of affinity

constants only depends on the relative rates at several substrate concentrations.

Protein-protein interactions between PKS components were discussed in the introductory

material to Chapter 2. In short, electrostatic and hydrophobic forces have been proposed to

determine ACP: partner protein interactions; in particular, negatively charged residues in

helix II of ACP have been appointed as the key players in ACP recognition by other

components of Type I and Type II PKS.

In this work, we have studied the interaction of ACP with KSβ by measuring the kinetic

parameters of decarboxylation of mutant malonyl-ACP by KSβ. All act mutants (except

E47V) achieved the same maximum velocity (kcat ~ 20 min-1), meaning that E47, E53 and

R72 are not involved in catalysis during initiation of polyketide synthesis. Notably, the


81

introduction of a bulky group in the E47V mutant severely impaired its suitability as a

substrate, suggesting a less-favoured docking position of E47V ACP onto KSβ. R72A

behaved essentially as the reference ACP. Remarkably, the same specificity constant kcat /

KM was measured for the reference act ACP and WT gris and dps ACP, in agreement with

previous reports showing successful complementation of Type II KSα/KSβ with

heterologous ACP.98,99

Also, we have provided further evidence to support the role of helix II as a recognition

motif in acyl carrier proteins: Michaelis constants of E47A, E47V and E53A ACP

increased three to four-fold when compared with act ACP (KM ~ 7-8 µM vs. 2-2.5 µM),

indicating less binding affinity of KSβ towards these mutants.

Our kinetic studies have also allowed us to achieve a better understanding of polyketide

biosynthesis by Type II PKS. In order to prime KSα with the acetate starter unit, two

possible mechanisms could be conceivable. First, an ‘internal transfer’ of acetyl-ACP

occurs directly from the KSβ active site to the KSα active site. The second scenario depicts

the release of acetyl-ACP into solution, and its subsequent encountering with KSα (Scheme

3.4). Whereas perhaps the first option would appear more effective, our results are

consistent with previous evidence that support the ‘external transfer’ of starter units,62,96 as

KSα is clearly able to load itself with acetate delivered by ACP from solution.

Nevertheless, our results cannot rule out the coexistance of the ‘internal’ and ‘external’

acetyl-ACP transfer pathways.


82

4. Chain elongation and cyclisation and release from the PKS

The biosynthesis of SEK4 and SEK4b by the actinorhodin minimal PKS in vitro composes

at least 20 different chemical reactions, including loading, initiation of polyketide

synthesis, chain elongation and cyclisation and release steps (see Scheme 1.18 in Chapter

1). Chapter 4 deals with the reactions catalyzed by KSα.

The first reaction catalyzed by KSα is its own priming with an acetate starter unit from

acetyl-ACP onto its active site cysteine, to form acetyl-S-KSα (Scheme 4.1.A). This occurs

via an acyl transfer step between the thiols of the PP arm in ACP and the active C169 in

KSα. Elongation of the polyketide chain occurs via decarboxylative Claisen condensations

between the acyl-S-KSα intermediate and malonyl-ACP (Scheme 4.1.B). After each

condensation, the growing polyketide chain must be transferred back to the active site of

KSα, again through an acyl transfer between the PP in ACP and KSα (Scheme 4.1.C).

It has been proposed by the Stanford group that, as the polyketide chain is elongated, it

extrudes into a ‘polyketide tunnel’ formed between KSα and KSβ (Section 1.2.3.4).31 This

may keep the reactive ketide groups far from each other, thus preventing spontaneous,

aberrant cyclizations. The polyketide tunnel can also provide an actual basis for the acyl

transfer from the terminal thiol of ACP to the active thiol in KSα, which must occur after

every condensation.

The acyl transfer between ACP and KSα has not been investigated in this work. We

have assumed that it quickly reaches equilibrium. This acyl transfer step as well as the

seven Claisen condensations have been considered here as the ‘elongation’ or ‘extension’

steps.

Finally, production of SEK4 and SEK4b must involve cyclization of the polyketide

chain and release of polyketides from the PKS into solution. It is unknown if and when the

cyclisation and release reactions are catalyzed by the ketosynthase complex (see Chapter 6

for further discussion).

KSα belongs to the thiolase superfamily of enzymes (see Section 1.2.3.3 in Chapter 1). The

kinetics of reactions catalyzed by other thiolases, such as the ketosynthases from Type I


83

and Type II FAS and other PKS, have been measured; and some examples are provided in

the following sections.

ACP

S

O

+ KS! KS"KS! KS"

Cys169S

O

KS! KS"

Cys169S

O

ACP

S

O+

O

KS! KS"

Cys169HS

ACP

S

O+

OHO

KS! KS"

Cys169HS

ACP

S

O+

O

KS! KS"

Cys169S

OOACP

SH

+

Scheme 4.1 Reactions catalyzed by KSα: A. Priming; B. Chain extension. C. Acyl transfer between the PP

thiol of ACP and the cysteine active site in KSα

4.1.1. Kinetics of FAS systems

Type I fatty acid synthases from mammals were the first fatty acid synthases studied

kinetically. In most cases, this was done by direct monitoring of the oxidation of NADPH

by spectrophotometry. For example, during the decades of the 70s and 80s, the Michaelis

constants for pigeon liver FAS (1.3 and 30 µM for acetyl and malonyl CoA,

respectively),159 bovine FAS (16.5 and 20 µM for acetyl-CoA and malonyl-CoA,

respectively),160 and chicken liver FAS (KM = 2.5 µM malonyl CoA)161 had been estimated.

Human FAS was studied later (KM = 6 µM malonyl CoA).162 Type I fatty acud synthases

from mycobacteria such as Mycobacterium smegmatis (KM =30 µM for acetyl CoA)163 and

Euglena gracillis (KM =1.3 and 5.1 µM for acetyl and malonyl CoA, respectively)164 were

also characterized. In summary, it appeared that the Michaelis constants ranged from 1 to

A.

B.

Acyl transfer: priming

extension

C.

Acyl transfer


84

30 µM for most of the fatty acid synthases studied. kcat values varied from 160 to 1200 min-1

in human and chicken liver FAS, respectively.161,162

Radioactive substrates have traditionally been used for the kinetic study of Type II fatty

acid synthases. The first Type II FAS studied was that of E. coli. KASIII exhibited a KM of

40-150 µM for acetyl-CoA, and 5-25 µM for malonyl-ACP.165,166 KASI and KASII showed

affinities of 30-80 µM for a variety of substrates, saturated and unsaturated C-12 to C-14

acyl-ACP.167

Other Type II systems have produced different kinetic parameters; for instance, KASIII

from Staphylococcus aureus (kcat = 16 min-1 and KM = 6.18 µM for acetyl CoA, and 1.76 µM

for malonyl-ACP)168 and KASI and KASII from Mycobacterium tuberculosis (kcat = 4.8

and 1.4 min-1 respectively, and KM = 13.5 µM for malonyl-ACP).169 The study of the plant

Type II FAS from Cuphea wrightii gave similar Michaelis constants (1.4 and 4.1 µM for

acetyl and malonyl CoA, respectively).170

4.1.2. Kinetics of Type III PKS

Type III polyketide synthases are very simple enzymes that catalyze repetitive

decarboxylative condensations between a starter unit and one or more molecules of malonyl

CoA (see Section 1.2.4 in Chapter 1). Therefore the kinetic study of these enzymes

provides a very useful model for other thiolases.

Type III polyketide synthases from plants and bacteria have been biochemically

characterized in vitro. Additionally, there is one Type III PKS from fungi that has been

kinetically studied.171 Although it accepted a wide range of starter units (and consequently

produced a variety of products), the most favourable substrate was stearoyl CoA 59 to

produce a pentaketide resorcylic acid 60 (Scheme 4.2). This proceeded with kcat = 3.1 min-1

and KM = 20.0 µM.171

S-CoA

O

15

OHO

O

OH

OH15

4 x malonyl CoA

Scheme 4.2 Reaction catalyzed by the pentaketide resorcylic synthase from Neurospora crassa

59 60

Type III PKS


85

Similar kinetic values had been reported for plant polyketide synthases. For instance, Jez,

Noel and co-workers had thoroughly studied the biosynthesis of chalcone by the Medicago

sativa (alfalfa) chalcone synthase (CHS). Using radioactive methods, they independently

measured the rate of decarboxylation of malonyl CoA by monitoring the conversion to

acetyl CoA, and the rate of product formation by counting radioactivity in thin layer

chromatography spots (Table 4.1).172

Decarboxylation of

malonyl CoA CHS assay (p-

coumaroyl CoA) CHS assay (malonyl

CoA) kcat (min-1 ) 4.1 2.2 3.7

KM (µM) 19.1 4.5 4.1

Table 4.1 Kinetic parameters for CHS.172

Abe, Noguchi and co-workers have reported similar kinetic values for the chalcone

synthases from Huperzia serrata (kcat = 2.0 min-1 and KM = 19.7 µM)173 and Scutellaria

baicalensis (kcat = 1.3 min-1 and KM = 36.1 µM).174 The same group has described the

activity of two Type III polyketide synthases from aloe (Aloe arborescens), a pentaketide

chromone synthase (PCS) and an octaketide synthase (OKS). While PCS uses 5 molecules

of malonyl CoA to afford 61, OKS produces the octaketides SEK4 19 and SEK4b 27 from

eight acetate units derived from malonyl CoA. These were less efficient enzymes working

at kcat = 0.4 and 0.1 min-1 and KM = 71 and 95 µM (PCS and OKS respectively). 175,176

OHO

OH O 61

The first bacterial Type III PKS to be characterized was RppA from Streptomyces

griseus.177 It catalyzes the formation of 1,3,6,8-tetrahydroxynaphtalene 62 from 5

molecules of malonyl CoA. Further characterization yielded similar kinetic parameters as

its plant relatives, with kcat = 0.77 min-1 and KM = 0.93 µM.178 Recently, a similar enzyme

has been found in Streptomyces coelicolor (kcat = 1.3 min-1, KM = 3.9 µM).179 The fastest

Type III enzyme has been found in the bacterium Pseudomonas fluorescens. PhID produces


86

phloroglucinol 63 from 3 molecules of malonyl CoA. A kcat value of 24 min-1 was

determined.180

OHOH

HO OH

OH

HO OH 62 63

4.1.3. Kinetics of Type I modular PKS (DEBS)

In vitro kinetic analysis of modular PKS was prompted by the discovery that individual

DEBS modules could be expressed and purified to homogeneity.181 Subsequent steady-state

kinetic analyses of DEBS modules were undertaken with engineered systems consisting of

a DEBS module supplemented with the TE domain.181,182 Using this strategy, the

conversion of the natural diketide substrate analogue, (2S, 3R)-2-methyl-3-

hydroxypentanoyl SNAC 64, to the corresponding triketide lactone 65 or 66 in the presence

of methylmalonyl CoA and NADPH was assessed by HPLC for modules 2 to 6 (Scheme

4.3).182 The catalytic constant kcat varied from 1.5 (module 3) to 9.5 (module 6) min-1, with

KM in the millimolar range (ca 4 mM).

O

O

O

O

OH

O

SNAC

OOH

HO S-CoA

OO

Scheme 4.3 Reactions catalyzed by A. Module 3 + TE; B. Modules 2, 5 and 6 + TE.

Reconstitution of the ‘natural’ ACP-bound substrates was performed by two methods. First,

by co-expression of the loading domain and module 1 with module 2, 5 or 6 + TE. Second,

by addition of the purified ACP domain and linker from module 4, which was

phosphopantetheinylated in vitro from the CoA analogue of 64.183 Both methods resulted in

64

65

66 + NADPH


87

a 100-fold decrease in the KM value with respect to the SNAC substrates, showing the

importance of the protic body of ACP in shuttling substrates to the PKS.

The Stanford group has also studied the performance of AT-KS didomains of modules

3 and 6 with the ACP from all six modules (termed ACP1 to ACP6 in Table 4.2).184 The

best ACP substrate for each didomain was that of the same module. For instance, ACP

from modules 1 and 6 were much worse substrates than ACP3 for the AT-KS of module 3;

and, in turn, ACP3 itself was not recognized efficiently by AT-KS from module 6. As

discussed in Chapter 2, the same group has recently described some of the structural basis

for this difference.92

AT-KS module 3 ACP1 ACP2 ACP3 ACP4 ACP5 ACP6 kcat (min-1) n.d. 0.03 0.18 0.21 0.16 n.d. KM (µM) n.d. 9 16 179 56 n.d. kcat / KM (min-1 mM-1) n.d. 3.2 11.0 1.2 2.8 n.d. AT-KS module 6 ACP1 ACP2 ACP3 ACP4 ACP5 ACP6 kcat (min-1) 0.005 0.012 n.d. 0.008 0.021 0.050 KM (µM) 16 31 n.d. 119 37 27 kcat / KM (min-1 mM-1) 0.28 0.39 n.d. 0.07 0.56 1.85

Table 4.2 Kinetic parameters for the reaction catalyzed by AT-KS didomains from modules 3 and 6 using

ACP from every module as a substrate.

4.2. Kinetics of the rate-limiting reaction catalyzed by KSα

We have used the methodology described in Chapter 3 to study the kinetics of the rate

limiting step within the reactions catalyzed by KSα. In Chapter 3 we showed that, if act

minimal PKS assays are supplemented with MCAT, then the kinetics of KSβ could be

studied. Addition of exogenous acetyl-ACP to MCAT-supplemented assays further

increased the rate of production of aromatic polyketides; in fact, saturation with acetyl-ACP

produced a 3-fold increase in rate (Section 3.1.2). Under these circumstances, reaction rates

depended linearly on the concentration of KSα/KSβ. In principle, this new rate limiting

step could be any of the intervening reactions, i.e. elongation of the chain catalyzed by

KSα, cyclisation or release. We first investigated whether under fast malonylation

conditions, and in the presence of excess acetyl ACP, chain extension or chain termination

steps (i.e. cyclization and release) were rate limiting.


88

4.2.1. Attempts to detect polyketide intermediates

In an attempt to establish whether chain extension or chain termination steps were rate

limiting, we searched for any polyketide intermediate prior to cyclization and release. If

only early intermediates could be observed, then that would suggest that elongation of the

chain was rate limiting. On the other hand, if a full octaketide prior to SEK4/4b formation

could be detected, then it would be more likely that cyclisation and release were slower

processes than chain elongation.

After each condensation, the resulting polyketide intermediate is transferred from the PP

arm in ACP to the active site cysteine in KSα (Scheme 4.1-C). In principle, polyketide

intermediates could be attached to either ACP or KSα, and the number of polyketide chains

under construction at any given time point was unlikely to significantly exceed the number

of active synthases. We thus conducted assays at high KSα/KSβ concentrations (3 µM),

hoping to observe part-completed acylated acyl carrier proteins or KSα by ESMS.

However, increasing KSα/KSβ concentrations also increased the overall rate of octaketide

production and malonyl CoA consumption, so aliquots of reaction mixtures containing

KSα/KSβ, holo-ACP, MCAT, acetyl-ACP and malonyl CoA were rapidly quenched by

addition of C4 resin,185 desalted and examined by ESMS. Only acetyl and malonyl ACP

species were observed (Figure 4.1). The detection of KSα by ESMS failed. This was

unfortunate, because, assuming that the acyl transfer step between the thiols of ACP and

KSα is in equilibrium (see introduction to Chapter 4), early polyketide intermediates most

likely are attached to KSα.

The same experiment was repeated and polyketides extracted and analyzed by HPLC to

search for triketides to heptaketides released into solution. We could only detect SEK4 and

SEK4b.

These experiments suggested no accumulation of polyketide intermediates either

attached to ACP (as judged by ESMS) or released into solution (as observed by HPLC).

This led us to speculate that the cyclisation and release steps are likely to be faster than the

elongation steps.


89

Figure 4.1 ESMS analysis of the ACP species in minimal PKS assays done in the presence of MCAT and

external acetyl-ACP. Expected masses: 9527 Da (malonyl-ACP), 9483 Da (acetyl-ACP).

4.2.2. Kinetics of the elongation step catalyzed by KSα .

In order to study the kinetics of KSα, we first tried to measure the kinetic constants for

malonyl-ACP as a substrate for KSα (i.e. the elongation step). The determination of kinetic

parameters in assays where excess acetyl-ACP was externally added was difficult due to a

range of circumstances. First, the rapid consumption of acetyl-ACP released holo-ACP into

solution. At 0.25 µM KSα/KSβ consumption rates of acetyl-ACP are ca. 12 µM min-1 under

saturating concentrations of malonyl-ACP (Section 3.1.2). In the presence of MCAT, the

generated holo-ACP was readily converted to malonyl-ACP. This introduced a degree of

uncertainty in the concentration of malonyl-ACP. Second, we assumed that the binding site

for acetyl-ACP onto KSα was the same as that for malonyl-ACP. Thus we anticipated that,

at high concentrations of acetyl-ACP and low concentrations of malonyl-ACP, we might

observe inhibition of the elongation reaction, due to the non-productive binding of acetyl-

ACP onto malonyl-ACP docking sites within KSα.

In an attempt to estimate the Michaelis constant for malonyl-ACP, we studied reaction

rates at fixed acetyl-ACP concentration (30 µM) and a range of malonyl-ACP

concentrations between 0.5 and 60 µM. When low malonyl-ACP concentrations were used,

we observed a lag in the production of polyketides which lasted 20-30 s. This agreed with

our predictions that acetyl-ACP was probably inhibiting polyketide synthesis when it was


90

in high excess of malonyl-ACP: during the lag, polyketide production was slower than

expected. As acetyl-ACP was consumed and malonyl-ACP concentration increased, the

inhibitory effect was attenuated causing an evident acceleration of polyketide production

(Figure 4.2). This pattern was reproducible at low (< 5 µM) malonyl-ACP concentrations,

however at malonyl-ACP higher than 10 µM the rate was constant during the first 1-2 min

of the reaction. For this reason it was not possible to measure KM for malonyl-ACP in the

reactions catalyzed by KSα.

Figure 4.2. Evolution of the production of polyketides with time at saturating acetyl-ACP concentration and

2.5 µM malonyl-ACP (triangles), 5 µM malonyl-ACP (circles) and 10 µM malonyl-ACP (crosses).

Therefore, in subsequent experiments we used high concentrations of malonyl-ACP (i.e. 10

times the KM values determined for the decarboxylation of malonyl-ACP by KSβ). We then

estimated the value of the catalytic constant by comparison of experiments done in the

presence and absence of acetyl-ACP (30 µM). kcat was determined as 58.2 ± 1.0 min-1

malonyl-ACP.

In order to gather more information about the interaction of ACP and KSα, we assessed

the effect of single mutations in ACP on the rate of reaction. Thus, acetyl-ACP-doped

assays were performed with the same act mutant ACP whose interaction with KSβ was

assessed in Chapter 3; as well as gris and dps ACP. Thus act acetyl-mutant-acyl carrier

proteins as well as gris acetyl-ACP and dps acetyl-ACP were produced and purified as


91

described for the reference act ACP. Polyketide production rates in the presence of excess

malonyl-ACP and the corresponding acetyl-ACP were then measured and compared with

control experiments lacking acetyl-ACP.

Thus holo-(mutant)-ACP concentration was set to 20 µM (reference act ACP and R72A,

gris and dps ACP) or 50 µM (E47A, E47V, E53A ACP). All assays (with and without

acetyl-ACP) were done at least in triplicate. kcat was around three times higher for each of

the act mutants (Table 4.3). Gris ACP and dps ACP behaved somewhat differently, and kcat

increased by a factor of 4.7 and 2.3 respectively. Higher concentrations of gris and dps

acetyl-ACP (up to 60 µM) did not produce further increase in rate.

ACP kcat (KSα) min-1

Increase with respect to KSβ

Act 58.2±1.0 2.8 E47A 56.3±4.0 2.8 E47V 10.6±0.5 3.0 E53A 62.3±2.0 3.6 R72A 71.3±2.1 3.5 Gris 45.1±2.3 4.7 Dps 38.4±4.2 2.3

Table 4.3 Catalytic constants in for a variety of ACP in the presence of excess acetyl-ACP.

4.3. Discussion

In Chapter 4 we have exploited the methodology developed in preceding chapters to

measure the kinetics of KSα. Thus we have used acetyl-ACP in high concentrations to

overcome the limitation in polyketide production rate imposed by KSβ (Chapter 3). Under

these conditions, the new rate limiting step could in principle correspond to either the

slowest of the reactions catalyzed by KSα, or the cyclization and release steps (see Scheme

1.18 in Chapter 1).

Here we hypothesizee that the elongation step catalyzed by KSα is the rate limiting step

in act minimal PKS in the presence of MCAT and exogenous acetyl-ACP, because SEK4

and SEK4b were the first isolable octaketides produced by the act minimal PKS. Attempts

to trap late polyketide intermediates either bound to ACP or released into solution failed.


92

On the other hand, there is evidence in the literature of early polyketide intermediates

attached to ACP189 or KSα.31 Taken together, this results suggested that the cyclization and

release steps proceeded at a higher rate than the chain elongation catalyzed by KSα.

First, we approached the study of the new rate limiting step by the determination of kinetic

parameters for malonyl-ACP as a substrate for KSα. When we assessed the rate of reaction

for a range of malonyl-ACP concentrations, we observed complex evolution of the rate as

the reaction proceeded, particularly during the first 20-30 sec. This we attributed to

inhibition of polyketide synthesis due to non-productive binding of acetyl-ACP to KSα,

when acetyl-ACP was in high excess of malonyl-ACP. This result evidences that the

docking site for acetyl and malonyl-ACP in KSα is the same.

Although these experiments could have been useful for estimating KM for acetyl and

malonyl-ACP in KSα-catalyzed reactions, the effect could not be studied further with the

methods developed in this work. In our steady state measurements, the first 5-10 seconds of

reaction could not be monitored, because the instrument required that time to start the

measurements. Future work should address the need for stopped-flow kinetic measurements

in order to study the reaction rate at low (< 5 µM) malonyl-ACP concentrations and a range

of acetyl-ACP concentrations. Under stopped-flow conditions, initial reaction rates can be

studied during the first milliseconds of reaction, when the pool of acetyl-ACP is still intact

and the concentration of malonyl-ACP corresponds to the concentration of holo-ACP

added.

Furthermore, radioactive acetyl-ACP could be used to learn the extent of acetyl starter

unit that is incorporated into products from exogenous acetyl-ACP, rather than from

decarboxylation of malonyl-ACP. This would be definitive evidence to assess if there is

any pathway for an ‘internal’ transfer of acetyl-ACP from KSβ to the active site of KSα

(Scheme 3.4). For that matter, these studies should be carried out with a KSα/KSβ complex

which is inefficient in catalyzing the initiation reaction (e.g. CA mutant – see Chapter 5).

We then decided to do assays under saturating concentrations of both acetyl-ACP and

malonyl-ACP. Reaction rates upon addition of acetyl-ACP depended on the nature of the

ACP. For example, act ACP supported a kcat value of ca. 60 min-1, whereas gris and dps

ACP were less effective (ca. 45 and 38 min-1 respectively). Interestingly, the reaction rate


93

in the presence of all act mutant-ACP experienced a 3-fold increase with respect to assays

in the absence of acetyl-ACP, i.e. in which the reaction catalyzed by KSβ is rate limiting.

For instance, act E47V ACP was 6 times worse a substrate than the reference act ACP in

KSβ, and also in KSα. This probably means that the binding of ACP onto KSβ and KSα is

very similar, and the introduction of a bulky valine residue has the same disadvantageous

effect on the catalysis of both enzymes.

The priming reaction of KSα with acetyl-ACP is similar to the priming of FAS with an

acetyl starter unit. Witkowski, Smith and co-workers studied the reaction rate of the

mammalian FAS in the absence and presence of added acetyl CoA.190 Interestingly, a 5-

fold increase in the reaction rate was also observed when exogenous acetyl-CoA was added

to their assays. Smith and co-workers suggested that, in the absence of added acetyl-CoA,

FAS exhibited a weak affinity for malonyl-ACP and decarboxylation of malonyl-ACP

occurred inefficiently. In contrast, in the presence of added acetyl-CoA, a stronger malonyl-

ACP binding site was created so that decarboxylation, coupled with condensation, took

place efficiently. They speculated that the β-ketoacyl synthase domain would exist in two

distinct conformations, one favouring the acylation of the active site cysteine and the other

favouring the decarboxylation of malonyl-ACP.

Likewise, act KSα/KSβ could exist in two different conformations, one favouring

decarboxylation of malonyl-ACP in the KSβ active site, and the other favouring the

acylation of the active site cysteine in KSα. This model would explain why the active site

of KSβ seemed to be buried in the crystal structure of KSα/KSβ published by the Stanford

group, which led them to dismiss the catalytic role of KSβ in the initiation of polyketide

synthesis: the published KSα/KSβ structure may represent a complex ready for extension,

rather than initiation. Indeed, electron density of intermediates attached to KSα was

observed in the Stanford crystal structure.

In this dynamic model, decarboxylation of malonyl-ACP by KSβ would trigger a

conformational change in the whole dimer to render a KSα that would then be in an ‘open’

conformation and ready to load itself with the acetyl starter unit. This model suggests that

the rate limiting step in the presence of exogenous acetyl-ACP is probably the priming of

KSα with an acetyl starter unit, because the rate of the KSα-catalyzed reaction seemed to


94

depend on the rate of malonyl-ACP decarboxylation by KSβ. This makes sense, because

KSα has to encounter acetyl-ACP in the bulk solution (see discussion to Chapter 3) in order

to catalyze the acetyl transfer from ACP onto its active site (Scheme 4.1.A).

The priming of KSα is analogous to the priming of Type III polyketide synthases with

the corresponding starter unit; also to that of DEBS modules primed with synthetic starter

units such as SNAC derivatives; and to that of priming of ZhuH KASIII-type initiation

ketosynthase in the Type II PKS of R1128 (see introduction to Chapters 3 and 4). Typical

kcat values for plant and bacterial Type III PKS range from 0.7 to 3.1 min-1. To date, the

fastest Type III enzyme characterized (PhID from the bacterium Pseudomonas fluorescens)

has a kcat value of 24 min-1. The DEBS Type I modular PKS catalyzes polyketide synthesis

at a similar rate with reported kcat values between 1.5 (module 3) and 9.5 (module 6) min-1.

The priming ketosynthase ZhuH is a faster enzyme, and catalyzes the condensation of

butyryl, propionyl or acetyl CoA with malonyl-ACP at 113, 74 and 58 min-1

respectively.131 These values are comparable to the catalytic constant determined here (kcat

~ 60 min-1).


95

5. Stoichiometric analysis of the act minimal PKS

The organization of a protein complex is defined at four levels. The amino acid sequence of

the protein is called its primary structure. In a folded protein, different regions form local

secondary structures, such as α-helices and β-sheets. The tertiary structure is formed by

packing such secondary structural elements into domains with defined topology, which, for

instance, can bring together amino acids far apart in the primary sequence. Finally, many

protein complexes exist as an arrangement of several polypeptide chains, same or different

from each other, which is known as the quaternary structure.95 In this case, the

stoichiometry of the complex is the relative abundance of each component polypeptide.

The quaternary structure is therefore the ultimate molecular organization of an enzyme

or complex of enzymes. In the latter case, it provides essential information about the

interaction and biomolecular communication among its different components.

Most enzymes of the thiolase superfamily exhibit a dimeric quaternary structure. For

example, in bacterial fatty acid synthases the ketosynthase typically exists in a dimeric form

(such as E. coli KASI, KASII and KASIII enzymes).48 Type III polyketide synthases are

also homodimeric, as are the ketosynthase domains of animal FAS85 and Type I modular

PKS.92 Other members of the thiolase superfamily, such as the biosynthetic thiolase from

Zoogloea ramigera, exist as tetramers.192

Initial studies by the Stanford group on the quaternary structure of the act KSα/KSβ

suggested that it had a tetrameric conformation in solution. Carreras and Khosla subjected

purified KSα/KSβ preparations to gel filtration chromatography, and found that the

complex eluted at an apparent molecular weight of ~168 kDa.53 Because KSα and KSβ

were co-purified as an equimolar mixture, this indicated that the major KSα/KSβ species

was probably the tetramer (which has an expected molecular weight of ca. 180 KDa).

Within the Cambridge and Bristol groups, a number of Ph.D students have also studied

the quaternary structure of KSα/KSβ. For example, gel filtration studies in the Cambridge

group also indicated that the predominant KSα/KSβ species eluted at the expected

molecular weight for the α2β2 tetramer.158 In the Bristol group, analytical

ultracentrifugation, gel filtration and electron microscopy studies also suggest that


96

KSα/KSβ exists mainly as a tetramer in solution (Dr. John Crosby, personnal

communication and ref. 120).

In preceeding chapters, KSα/KSβ complexes have been represented simply as a dimer. In

order to study the stoichiometry of the act minimal PKS, we may now consider that the

dimeric and tetrameric forms of KSα/KSβ are in equilibrium (Scheme 5.1), and that the

major KSα/KSβ species in solution seems to be the tetramer.

KS! KS"

KS! KS"

KS! KS" KS! KS"+

Scheme 5.1 Dimer-tetramer KSα/KSβ equilibrium.

There is little evidence about the stoichiometry of ACP: KSα/KSβ association. In the

Bristol group, tryptophan fluorescence experiments were performed to address the question

of how many acyl carrier proteins bound each KSα/KSβ complex. ACP titrations in

KSα/KSβ solutions suggested a 1:1 stoichiometry relation between ACP and KSα/KSβ.120

Although these analytical methods are of course very useful, they only provide information

about the state of proteins in solution, not about the catalytic activity of the enzyme

complexes. It seemed necessary to study and characterize these protein complexes under

conditions upon which they were active, and polyketides were produced in an effective

manner. Thus we set ourselves to investigate the relationship between the catalysis and the

quaternary structure of KSα/KSβ and ACP: KSα/KSβ stoichiometry, using the quantitative

methods described in previous chapters of this thesis.

5.1. Stoichiometry of the KSα /KSβ complex –assays with mutant KSα /KSβ

The study of the stoichiometry of KSα/KSβ in solution is intrinsically difficult due to the

protocol we use to purify them. KSα contains an N-terminal hexa-histidine tag which


97

allows its purification by nickel-affinity purification.8 Because KSα and KSβ share

extensive surface area,31 KSβ is not washed away during the affinity chromatography

procedure; in other words, KSβ is co-purified in KSα preparations. Therefore it is not

feasible to vary the relative concentrations of the ketosynthase components in order to

study their respective kinetic properties. Previous attempts within the Bristol group to

express and purify soluble and active KSα and KSβ as individual proteins, have been

unsuccessful to date.142

The Bristol and Cambridge groups had previously studied the activity of a series of

KSα and KSβ mutant proteins, namely CA, AQ and AA mutant KSα/KSβ complexes (see

introduction to Chapter 3).62 A combination of HPLC analysis of polyketides as well as

ESMS analysis of acyl-ACP species, suggested that a CA KSα/KSβ complex, in which the

glutamine active site of KSβ had been mutated to alanine, was deficient in decarboxylation

of malonyl-ACP to acetyl-ACP.62 Conversely, an AQ KSα/KSβ complex was an efficient

decarboxylase of malonyl-ACP, as demonstrated by detection of acetyl-ACP by ESMS.62

Using this qualitative method, it was estimated that AQ KSα/KSβ decarboxylated malonyl-

ACP at approximately 40 µM h-1. Acetyl-ACP most likely was released into solution, rather

than transferred ‘internally’ to KSα (see introduction to Chapter 3 and Section 3.3).

Therefore we speculated that addition of AQ KSα/KSβ to act minimal PKS assays

using the wild type KSα/KSβ (i.e. CQ KSα/KSβ), would effectively increase the relative

concentration of KSβ (Scheme 5.2). If this hypothesis held, then we would be able to fix

the concentration of KSα (by fixing CQ KSα/KSβ concentration), and increase the

concentration of KSβ simply by increasing AQ KSα/KSβ concentration in PKS assays.

QCQA

ACP

S

HO

O

O

ACP

S

O

Polyketide synthesis

Scheme 5.2 Hypothetical use of AQ KSα/KSβ to increase the rate of KSβ-mediated decarboxylation of

malonyl-ACP. KSα (red) and KSβ (green) are depicted by their corresponding active sites (see text).


98

We decided to test this hypothesis, i.e. that the relative KSβ concentration could be

‘increased’ by adding AQ KSα/KSβ to act minimal PKS assays. Thus, we used the wild

type (CQ) KSα/KSβ (0.25 µM) in the presence of saturating concentrations of malonyl-

ACP (i.e. holo-ACP, malonyl CoA and MCAT), and we varied the concentration of AQ

KSα/KSβ. The highest PKS activity was reached at AQ KSα/KSβ concentrations of 4 µM

and higher. At saturating concentrations of AQ KSα/KSβ, an increase in kcat of only 1.3-

fold was measured, from 20.6 to 26.8 min-1 (Figure 5.1). This compares with a value of

58.2 min-1 as determined in Section 4.2.2 when excess acetyl-ACP was added to the

minimal PKS.

Figure 5.1 Rate of polyketide production by the act PKS (supplemented with MCAT) using CQ KSα/KSβ

(0.25 µM) at a range of AQ KSα/KSβ concentrations.

Thus it appeared that the presence of AQ KSα/KSβ could not substitute the external

addition of acetyl-ACP. It was conceivable that AQ KSα/KSβ was using malonyl-ACP as a

substrate thus decreasing its concentration, and producing high concentrations of acetyl-

ACP, which may inhibit the PKS (as shown in Section 4.2.2). To test for this hypothesis,

we added acetyl-ACP (30 µM) to these assays, and measured the reaction rate. kcat was

determined as 61.0 min-1, very similar to the value of kcat of the native PKS in the presence

of acetyl-ACP. Therefore the hypothesis that the acetyl-ACP produced by AQ was

inhibiting the PKS did not hold.


99

Another possibility was that our AQ KSα/KSβ preparation was not active. We decided to

test for this hypothesis using another system; and we purified the CA KSα/KSβ mutant

complex, i.e. a KSα/KSβ complex which is defective in decarboxylation, because the

change in rate should be even more dramatic.

5.1.1. Kinetic study of a mutant CA KSα /KSβ complex

In the light of the continuous method we developed in Chapter 3 to measure the kinetics of

KSβ, we decided to study the activity of the CA KSα/KSβ complex in depth. Thus

production of polyketides by CA KSα/KSβ was assessed in the presence of malonyl CoA,

MCAT and a variety of holo-ACP concentrations. Initial reaction rates were then measured

(Figure 5.2). Kinetic parameters of kcat = 2.2 ± 0.3 min-1 and KM = 2.00 ± 0.66 µM malonyl-

ACP were determined, i.e. the CA KSα/KSβ exhibited the same affinity as the CQ

KSα/KSβ for malonyl-ACP, but could only function ca. 10 times slower than the wild type

protein.

Figure 5.2 Kinetics of the decarboxylation of malonyl-ACP by CA KSα/KSβ. A. Plot of reaction rate vs.

substrate concentration and hyperbolic fit. B. Hanes linear plot of the rate data.

Following the methodology developed in Chapter 3, we then turned our efforts to

complement this decarboxylase-deficient CA KSα/KSβ by addition of extra starter unit,

acetyl-ACP. We anticipated that addition of external acetyl-ACP would surpass the lack of

decarboxylation activity of the CA KSα/KSβ. Therefore we expected to observe similar

reaction rates as measured with the wild type CQ KSα/KSβ (kcat ~ 60 min-1). Surprisingly,


100

when acetyl-ACP (10-60 µM) was added to CA KSα/KSβ assays, reaction rates increased

only 3-fold, up to 6.4 min-1, instead of the expected 30-fold to reach WT levels of activity.

The initial reason for purification of the CA KSα/KSβ complex was to complement this

decarboxylation-defective protein complex with the acetyl-ACP generated by AQ

KSα/KSβ. This could tell us whether or not our AQ KSα/KSβ preparation was active and

efficiently decarboxylated malonyl-ACP.

Thus we performed minimal PKS assays using the CA KSα/KSβ in the presence of

saturating concentrations of malonyl-ACP, and varied the concentration of AQ KSα/KSβ

complex. Again, we regarded these assays as experiments in which KSα concentration (i.e.

CA KSα/KSβ concentration) was fixed whereas KSβ concentration (i.e. AQ KSα/KSβ

concentration) was varied. Titration of AQ in these assays indicated saturation of CA

KSα/KSβ with the acetyl-ACP produced by AQ, at AQ concentrations higher than 2 µM

(Figure 5.3). Under these circumstances, kcat was determined as 6.0 min-1, which compares

well with 6.4 min-1 (kcat in the presence of excess acetyl-ACP). So it appeared that AQ was

active, and was capable of producing enough acetyl-ACP at least to saturate CA KSα/KSβ.

Figure 5.3 Rate of polyketide production by the act PKS (supplemented with MCAT) using CA KSα/KSβ (1

µM) at a range of AQ KSα/KSβ concentrations.

Surprisingly, when we added exogenous acetyl-ACP (30 µM) to assays in which CA

KSα/KSβ was complemented with the highest concentrations of AQ KSα/KSβ, further


101

increase in rate was observed. In these assays kcat reached 9.6 min-1. This result was

unexpected, because it meant that the presence of AQ KSα/KSβ somehow increased the

activity of CA KSα/KSβ (but not the wild type CQ KSα/KSβ), (Table 5.1) in the presence

of saturating concentrations of acetyl-ACP.

Addition (saturating concentrations)

kcat WT KSα /KSβ (min-1)

kcat CA KSα/KSβ (min-1)

none 20.6 ± 0.9 2.2 ± 0.3

AQ 26.8 ± 4.0 6.0 ± 1.0

Acetyl-ACP 58.2 ± 1.0 6.4 ± 0.7 AQ and acetyl-ACP

61.0 ± 4.8

9.6 ± 1.0

Table 5.1 Summary of kcat values for WT and CA KSα/KSβ under a range of conditions.

5.2. Stoichiometry of the ACP: KSα /KSβ complex – inhibition of KSα/KSβ by apo-

ACP.

Inhibition studies can provide important information about the stoichiometry of protein

complexes. The general mechanism for enzyme inhibition describes two possible types of

behaviour. An inhibitor which is analogous to the substrate may only bind to the free

enzyme, and this would suggest that there is only one binding site for the inhibitor (as well

as for the substrate to which the inhibitor is analogous). In this case, the inhibition is termed

‘competitive’ (as substrate and inhibitor compete for the same binding site on the enzyme)

and it is characterized by a competitive inhibition constant, Kic (Scheme 5.2).94

On the other hand, an uncompetitive inhibitor only binds to the enzyme-substrate

complex, and it does so with an affinity described by an uncompetitive inhibition constant,

Kiu (Scheme 5.2).94 In this scenario, the inhibitor does not compete with the substrate,

which suggests that inhibitor and substrate bind to different sites on the protein.

Finally, if the inhibitor binds to both the free enzyme and the complex formed between the

enzyme and the substrate, then this may be evidence for multiple substrate binding sites. In

this situation, the enzyme would be able to bind (at least) two substrates at the same time.


102

Apo-ACP is the inactive form of ACP that lacks the PP arm (see Section 1.2.3.1). NMR

studies of act apo-ACP and malonyl-ACP suggest that both species have very similar

structures in solution.193 Therefore apo-ACP was a good entry point in order to perform

inhibition studies with the act PKS. This could be detected by a decrease in polyketide

production rates.

We expected that apo-ACP would at least compete with malonyl-ACP for binding sites

on KSα/KSβ (Scheme 5.3). For instance, apo-ACP could compete with malonyl-ACP for

binding sites only on KSβ; or with acetyl and/or malonyl-ACP for binding sites only on

KSα. Competition could also occur in both subunits, KSα and KSβ. In these three

situations, only competitive inhibition would be observed. The presence of an

uncompetitive component in the inhibition pattern would be evidence for an allosteric

relation between the catalysis of KSα and KSβ.

KS!/KS" + malonyl-ACP KS!/KS". malonyl-ACP polyketide production

apo-ACP

+

KS!/KS". apo-ACP

+

KS!/KS". apo-ACP. malonyl-ACP

KicKiu

+ malonyl-ACP polyketide production??(see discussion)

apo-ACP

Scheme 5.3 General mixed inhibition mechanism for apo-ACP as an inhibitor of KSα/KSβ

Act apo-ACP was produced by expression in E. coli and purified essentially as holo-ACP.

The yield was 30-40 mg L-1. When apo-ACP was incubated with malonyl CoA and

KSα/KSβ, a background rate of polyketide production was observed, which must be

attributed to the presence of minor amounts of holo-ACP formed by

phosphopantetheinylation of apo-ACP by E. coli ACPS, although the presence of holo-

ACP was not observed by ESMS (Figure 5.1). By comparing the reaction rate in assays

with apo-ACP, with our previous kinetic results using holo-ACP (Chapter 3), it was

estimated that 0.5-1% of the total ACP concentration was holo-ACP.


103

Figure 5.4 Mass spectrum of act C17S apo-ACP (expected, 9101 Da, also observed as the sodium and

potassium adduct at 9124 and 9140 Da, respectively).

We first used the α-ketoglutarate dehydrogenase assay (Section 2.4.1) to test for MCAT

inhibition by apo-ACP. Thus, MCAT (2 nM) was used to catalyze the malonylation of S.

coelicolor FAS ACP (10-20 µM, kindly donated by Eliza Ploskon) and act apo-ACP was

added in high concentration (200 µM). No inhibition of MCAT activity was observed.

Then we tested the ability of apo-ACP to inhibit KSα/KSβ. Thus, KSα/KSβ (0.25 µM)

was incubated with holo-ACP, malonyl CoA, MCAT (0.5 µM) and a range of

concentrations of apo-ACP. We observed reduced reaction rates with increasing apo-ACP

concentrations. We then executed a full kinetic study of this inhibitory effect. Apo-ACP

was determined to be a mixed inhibitor of KSα/KSβ with inhibition constants Kic = 50 ± 12

µM and Kiu = 137 ± 28 µM (Figure 5.5). Interestingly, no deviations from the linear

inhibition pattern were observed (see discussion).

Figure 5.5 Inhibition of KSα/KSβ by apo-ACP at holo-ACP concentrations of 2.5 µM (full squares), 5 µM

(circles), 7.5 µM (squares), 15 µM (full circles) and 25 µM (triangles).

A. B.

Kic Kiu


104

5.3. Discussion

The decarboxylation of malonyl-ACP by KSα/KSβ had previously been assessed by

qualitative ESMS analysis of the acetyl-ACP generated by mutant KSα and KSβ proteins

in which the corresponding cysteine (C169) and glutamine (Q161) active sites had been

mutated to alanine.62 The underlying assumption was that mutation of the respective active

sites completely abrogated the activity of KSα or KSβ towards decarboxylation of

malonyl-ACP, while leaving the activity of the other component unaffected. Here we

decided to test this hypothesis.

First, we studied the effect of ‘increasing’ KSβ concentration in the system by adding

AQ KSα/KSβ to PKS assays in which either CQ or CA KSα/KSβ was used. In Chapter 3

we showed that external addition of acetyl-ACP to act minimal PKS assays caused a 3-fold

increase in rate (kcat varied from 20.6 to 58.2 min-1), thus meaning that acetyl-ACP was the

limiting species in the presence of excess malonyl-ACP (Section 3.1.2). By adding AQ CQ

KSα/KSβ to the assays, we expected the same increase in rate due to AQ-catalyzed

production of acetyl-ACP, however, we only observed a 1.3-fold increase.

We then supplemented CA KSα/KSβ with AQ KSα/KSβ. The reaction rate under these

conditions corresponded to that observed under saturation of KSα/KSβ with external

acetyl-ACP. We expected that the addition of external acetyl-ACP (or AQ KSα/KSβ for

that matter) to assays using CA KSα/KSβ would surpass the lack of a functional KSβ and

thus an increase of 30-fold in the reaction rate would be observed, i.e. back to WT levels of

activity. Surprisingly, when extra acetyl-ACP (or AQ KSα/KSβ) was added to CA

KSα/KSβ assays, the increase in rate was only three-fold. This meant either that CA

KSα/KSβ was not active, or that the lack of a functional KSβ in the ketosynthase complex

somehow impaired the catalytic activity of CA KSα/KSβ.

Unexpectedly, addition of external acetyl-ACP to this assay (i.e. to minimal PKS assays

using CA KSα/KSβ and in the presence of saturating concentrations of AQ KSα/KSβ)

produced further increase in rate and kcat raised to 9.6 min-1. This was an important result,

as it suggested that CA KSα/KSβ was damaged at yet another stage than just the initial

decarboxylation of malonyl-ACP. This also showed that KSβ affected the rate of the

reactions catalyzed by KSα, which is in agreement with our results using the wild type

KSα/KSβ and mutant acyl carrier proteins (see discussion to Chapter 4), where we


105

observed a dependence of the rate of KSα-catalyzed reactions on the rate of the initiation

step catalyzed by KSβ.

The interpretation of these results requires consideration of the quaternary structure of

KSα/KSβ. Let us suppose that the active KSα/KSβ species is the dimer of heterodimers,

(i.e. α2β2 tetramer), as suggested by the early work of Khosla53 and ongoing research in the

Bristol group (Scheme 5.1).120

In this case, the addition of AQ KSα/KSβ to PKS assays would affect the composition

of KSα/KSβ tetramers. Assuming the equilibrium constant K = 1, and for concentrations of

CQ and CA KSα/KSβ of 0.25 and 1 µM respectively, > 95% of the total enzyme capable of

sustaining polyketide production would be in complex with AQ at AQ KSα/KSβ

concentrations higher than 5 µM (Scheme 5.4).

Q

C

C

Q A

A

Q A

C

Q

+K = 1Q Q

C

C

A A

A

Q A

C

Q

+K = 1Q AA

2 x

2 x

Scheme 5.4. The addition of AQ affects the composition of A. CQ KSα/KSβ tetramers; B. CA KSα/KSβ

tetramers.

Our results could be explained if the active KSα/KSβ species is indeed tetrameric and, as

speculated in Chapter 4, the catalysis of KSα and KSβ is accompanied by a conformational

change. For example, in assays using CA KSα/KSβ, the species in solution would be

{CA|CA}. In the presence of AQ KSα/KSβ, the major species with PKS activity would be

{CA|AQ}, (Scheme 5.4). The species {CA|AQ} was found to be more active than

{CA|CA}: the presence of an active KSβ favoured the catalysis of the tetramer at

saturating concentrations of acetyl-ACP (i.e. when acetyl-ACP was externally supplied).

This implicated the presence of Q in the active site of KSβ in one dimer in improving the

catalytic behaviour of KSα in the other dimer.

A.

B.


106

Thus, the generation of an active KSβ in the complex {CA|AQ} probably enhances the

catalysis of the tetramer by a different means than production of acetyl-ACP as a substrate

for KSα (because the system is saturated with acetyl-ACP). The active KSβ may prompt a

more favoured conformational state in its KSα counterpart. Thus a conformational change

in an active KSβ subsequently would affect the whole structure of the tetramer.

Furthermore, this is consistent with our observations on the supplementation of CA

KSα/KSβ complex with external acetyl-ACP, when we found that the reaction rate

increased only 3-fold, and not 30-fold to reach WT levels. If the role of KSβ were only the

decarboxylation of malonyl-ACP to acetyl-ACP, then CA KSα/KSβ should perform at ~ 60

min-1 in the presence of excess acetyl-ACP. However, the 10-fold decrease in

decarboxylation activity (kcat changes from 20.6 to 2.2 min-1) of KSα/KSβ due to the

substitution of the KSβ active site glutamine for alanine probably renders a ‘static’ KSβ,

which lacks the conformational flexibility that facilitates the catalysis of KSα. As a result,

saturating concentrations of acetyl-ACP produced an increase in kcat from 2.2 only to 6.4

min-1 (far from WT levels at 60 min-1).

In the presence of excess malonyl-ACP, production of acetyl-ACP by AQ KSα/KSβ should

depend linearly on the concentration of AQ KSα/KSβ. A re-plot of the data in Figures 5.1

and 5.3 shows a linear increase in polyketide production reaction rate with AQ KSα/KSβ

concentration at AQ KSα/KSβ concentrations lower than 2 µM (Figure 5.6). This is most

likely due to increasing acetyl-ACP concentrations. However, at AQ KSα/KSβ

concentrations higher than 2 µM, the linear relationship is not maintained.

We think this is due to lack of decarboxylation activity of the AQ KSα/KSβ complex

when compared to the wild type. As KSα in AQ KSα/KSβ complexes is not active, it also

lacks the conformational flexibility which is necessary for an efficient catalytic activity of

the tetramer. It may be possible, for instance, that the absence of a conformational change

in KSβ leads to product inhibition. In this case, acetyl-ACP (the product of the reaction

catalyzed by KSβ) may decrease the activity of KSβ, when acetyl-ACP concentrations

reach certain threshold. This would render inactive AQ KSα/KSβ complexes. This may

explain why, for example, in assays using the wild type CQ KSα/KSβ, the increase in rate

due to addition of AQ KSα/KSβ was only 1.3-fold, instead of 3-fold to reach the level of

activity when CQ KSα/KSβ is saturated with acetyl-ACP.


107

Figure 5.6 Re-plot of data in Figures 5.1 and 5.3.Titration of AQ in assays performed with WT KSα/KSβ

(blue, 0.25 µM concentration) and CA (red, 1 µM concentration).

5.3.1. Stoichiometry of ACP: KSα /KSβ complexes

Inhibition of FAS and PKS by inactive forms of ACP has previously been reported. For

example, the group of Cronan reported a decrease in cell growth when FAS apo-ACP was

overexpressed in E. coli. They further showed that apo-ACP inhibited the enzyme sn-

glycerol-3-phosphate acyltransferase in vitro, resulting in the inability to transfer the mature

fatty acid to sn-glycerol-3-phosphate.194 Another example of ACP inhibition of a

ketosynthase was observed in the R1128 PKS system. The apo form of ACP was reported

to inhibit the priming ketosynthase, ZhuH.13 Also, the Stanford group reported that act apo-

ACP was a competitive inhibitor of KSα/KSβ with a Kic = 13 µM.53 However, this study

was done using holo-ACP from the frenolicin PKS. Furthermore, it was not ruled out that

the inhibitory effect observed was in fact due to apo-ACP inhibition of MCAT rather than

of KSα/KSβ.

The study of the inhibition of MCAT and KSα/KSβ by apo-ACP (Section 5.2) showed that

apo-ACP did not inhibit MCAT, but was a mixed inhibitor of KSα/KSβ. This suggested

that there are two different binding sites for ACP in the KSα/KSβ complex. In effect, the

general scheme for mixed inhibition (Scheme 5.3) envisages an uncompetitive inhibition


108

component due to the binding of the inhibitor to the enzyme-substrate complex. As

KSα/KSβ binds malonyl ACP for two different reactions (initiation and extension) it

seemed likely that the observed pattern of inhibition represented apo-ACP binding at the

extension (i.e. KSα) and initiation (i.e. KSβ) sites. Kic for inhibition by apo-ACP was 50

µM, a factor of 20-fold higher than KM for malonyl ACP as a substrate (2.39 µM). This

difference suggested that KSα/KSβ recognizes malonyl-ACP preferentially, meaning either

a specific binding interaction to malonyl-phosphopantetheine, or that malonyl-ACP takes

up a different conformation to apo-ACP. The latter idea is not supported by structural

studies but it may still be valid because CoA, which possesses the same

phosphopantetheine moiety as holo-ACP, does not appear to inhibit this step (see Section

2.5 in Chapter 2).

The inhibition behaviour shown in Figure 5.5 corresponds to the classical pattern of a

mixed inhibitor: linear relationships between the inverse of the reaction rate and the

inhibitor concentration. Within the range of concentrations studied, no deviations from this

linear behaviour were observed. This can also be useful for discussion and interpretation of

results.

Cooperativity is a property of multimeric enzymes that results from ‘cooperation’

between different active sites. It can either be ‘positive’, when substrate binding on one

active site increases the activity of neighbouring sites (i.e. it increases the affinity of the

enzyme towards the substrate); or ‘negative’, when the activity of neighbouring sites is

decreased upon substrate binding to one site. Cooperative effects can be detected from

inhibitions studies. They usually appear as deviations from the linear behaviour in the

inhibition plots described above.

Many enzymes that show allosteric regulation are also cooperative. We have

hypothesized in Chapters 4 and 5 that a conformational change in KSβ produces an

allosteric modification in KSα. If this was essential for efficient PKS activity of KSα/KSβ,

then cooperative effects between the active sites of KSα and KSβ could also occur. For

example, if binding of apo-ACP onto KSβ produced a change in KSα conformation, then a

change in the affinity of KSα towards ACP could be expected. However, the study of

inhibition of KSα/KSβ by apo-ACP did not support this idea because only linear

relationships between the inverse of the rate and the inhibitor concentration were observed.


109

This suggests that the conformational change that renders active KSα occurs as a result of

malonyl-ACP decarboxylation, rather than upon substrate binding onto KSβ.

Deviations from the linear behaviour in inhibition patterns are not only a sign of

cooperative effects. They also point towards hyperbolic inhibition.94 In Scheme 5.3, the

intermediate {KSα/KSβ. apo-ACP. malonyl-ACP} may or may not be a dead-end species.

If this species were still capable of producing polyketides, then apo-ACP would be a

hyperbolic inhibitor of KSα/KSβ and the linear relationships between the inverse rate and

inhibitor concentration would not be maintained.94

Assuming that KSα and KSβ have indeed two different binding sites for ACP, then

there are two possibilities to yield such {KSα/KSβ. apo-ACP. malonyl-ACP} species: first,

that apo-ACP binds to KSα; second, that apo-ACP binds to KSβ. Interestingly, we only

observed linear relationships between rate and inhibitor concentration. These results

suggest that both possibilities render inactive KSα/KSβ complexes, incapable of polyketide

synthesis. It is clear that if apo-ACP binds KSα, then PKS activity is inhibited. It appears

that polyketide synthesis is also inhibited if apo-ACP binds KSβ. Again, this could support

our hypothesis of the conformational change: if apo-ACP binds KSβ, it may prompt a

conformational flip in KSβ that probably renders a KSα/KSβ complex ready for

decarboxylation of malonyl-ACP, rather than for polyketide synthesis.

Future work on the study of ACP: KSα/KSβ complexes should investigate whether or not

KSα and KSβ have indeed two different binding sites for ACP. A simple experiment could

illustrate whether apo-ACP inhibits KSα, KSβ or both: inhibition assays could be

performed in the presence of excess acetyl-ACP. If no decrease in rate could be detected,

then it would be clear that apo-ACP does not inhibit the reaction catalyzed by KSα. The

opposite would hold if polyketide production rates decreased with increasing apo-ACP

concentration. Comparison of these experiments with the assays performed in this work

could identify which ketosynthase subunit is inhibited by apo-ACP.


110

6. Kinetics of an extended act minimal PKS

In preceding chapters the production of the octaketides SEK4 19 and SEK4b 27 in vitro by

the actinorhodin minimal polyketide synthase from malonyl CoA was studied. Although

WT S. coelicolor A3 (2) does indeed produce some SEK4 and SEK4b (Dr. Greg Challis,

personal communication), it is generally assumed that, in vivo, the hypothetical octaketide

intermediate 18 is mostly processed to the final product actinorhodin 9 by the remaining

components of the act polyketide synthase. Early modifications of 18 include

ketoreduction, aromatization and cyclization (see Scheme 1.3 in Chapter 1).70

Identification of the gene product responsible for ketoreductase activity in the act cluster

was initially approached by in vivo experiments. Bartel, Keller and collaborators used S.

galilaeus 31671, which produces the polyketide 9-hydroxy-aklavinone 67 from a

propionate starter unit and 10 malonate extender units, as a host for genetic

manipulation.196 Transformation of S. galilaeus 31671 with a plasmid that contained the

actIII gene resulted in production of 68 (Scheme 6.1). This suggested that actIII encoded a

ketoreductase that catalyzed the reduction of C-9 of the hypothetical intermediate 69,

resulting in the formation of a secondary alcohol (the stereochemistry of the act KR-

catalyzed reduction has not been determined to date).32

O O O

O

O O O O

COOH

O

O O O

HO

O O O O

COOH

O99

OCOCH3

OH

OHOH

O

OOH

HO

OCOCH3

OH

OHOH

O

OOH

Scheme 6.1 Complementation of S. galilaeus with act KR resulted in production of a reduced polyketide (the

propionate starter unit is shown in red).196

act KR

S. galilaeus S. galilaeus

67 68

69


111

In vivo experiments with S. coelicolor A3(2) mutants also suggested that the necessary KR

activity was provided by actIII. Using an actVII S. coelicolor mutant which was able to

produce polyketides (but not actinorhodin), Zhang, He and co-workers isolated and

characterized mutactin 20.197 Mutactin was hypothesized to arise from reduction of C-9 of

the intermediate 18. The corresponding reduced octaketide 70 then cyclised to form

mutactin 20, which exhibited the same cyclisation pattern as 19 and indeed actinorhodin 9

in the formation of the first ring between the carbons C-7 and C-12 (Scheme 6.2).

S-ACP

OOOO

O

O OO

HO

O

O

O

OH

O

OHO

O

OH

OH

HO

O

O

OHO

O

OH

HO

O

S-ACP

OOOO

HO

O OO

99

7

7

12

12 12

12

7

10

10

15

15

7

Scheme 6.2 Effect of addition of KR to act minimal PKS in in vivo experiments.

Further in vivo studies using S. coelicolor CH999 as the host for transformation with the

pRM5 vector (see Section 1.2.2.1) showed that, in the absence of KR, a second product of

the act minimal PKS was SEK4b 27, which originated by an alternative cyclisation of the

octaketide backbone 18, i.e. between carbons C-10 and C-15.198 As mutactin 20 was the

major product when KR was co-expressed with the act minimal PKS,199 a second role for

KR was assumed to be the control of the regiospecificity of the first cyclisation.200

All Type II PKS-derived polyketides that undergo ketoreduction are only reduced at the

ninth carbon from the carboxy terminus of the assembled polyketide. This strict

regiochemistry is independent from polyketide chain length (octaketide to decaketide) and

the nature of the starter unit.200 In vivo experiments could not explain this regio-

especificity, nor could they identify the substrate for act KR: was it the full-length

18 70

19 27 20

KR

Cyclisation Cyclisation


112

octaketide 18 (as assumed above)196 or an earlier intermediate? Furthermore, if KR used an

octaketide as a substrate, which was the timing of the first cyclization (i.e. before or after

reduction)?

The role of KR in directing the first cyclization was evidenced by work by Kunnari,

Hilionko et al, using S. galilaeus 31365 mutants for metabolite analysis studies.201 S.

galilaeus 31365 produces aklavinone 71 (which shows reduction at C-9, and C-7 to C-12

cyclization pattern analogous to mutactin) from a propionate starter unit and 9 malonate

extender units. The mutant S. galilaeus 31365 HO61 produces 72, which results from a

C10-C15 first cyclization (Scheme 6.3), analogous to that of SEK4b. When this mutant was

transformed with a plasmid carrying a gene capable of providing KR activity, formation of

72 was abrogated, and production of aklavinone 71 was recovered (Scheme 6.3).

Since the ketoreduction changes the hybridization of the substrate carbonyl (from sp2 to

sp3), it introduces a defined bend of the poly-β-keto intermediate chain. Thus, Kunnari,

Hilionko et al speculated that reduction occurred when the polyketide chain was complete,

but before the first cyclization. In this model, the chain, once reduced at C-9, takes up a

favoured orientation for the subsequent aldol condensation between C-7 and C-12.201

Alternatively, one could argue that KR may prompt a conformational change in the PKS

complex, rendering a more rigid struture that prevents the C-10 to C-15 cyclization.

O O O

O

O O O O

COOHO

O O O

O

O O O O

COOH

O9

10

OH

O

OOH

HO

OCOCH3

OH

OHOH

O

OOH

7

12

12

7

O

OH

O10

15

15

72 71

Scheme 6.3 Production of 72 and 71 by S. galilaeus HO61 and S. galilaeus HO61 complemented with KR,

respectively. The propionate starter unit is shown in red.

S. galilaeus HO61

S. galilaeus HO61 + KR


113

An alternative model, in which the first cyclization occurs prior to reduction was proposed

by Korman, Tsai and co-workers, who constructed a series of in silico models of substrate

binding to act KR, and speculated that the first cyclisation must occur prior to interaction of

the polyketide with KR.202 This model has been recently supported by experimental

evidence. The group of Tsai studied the activity of act KR in vitro with a series of possible

substrates, 74 to 81.203 They could not detect activity of KR towards the products 77 to 81,

or indeed towards acetyl CoA or acetoacetyl CoA. On the other hand, the bicyclic

compounds 74, 75 and 76 were used by KR as a substrate with unequal specificity (Table

6.1).

HO

H

H

H

OO

O

O

O

OO

O

O

O

O

O

O

O

O

79 80 81

Substrate kcat (s-1) KM (mM) kcat / KM (s-1 mM-1)

74 2.6 0.79 3.2 75 0.16 3.9 0.04 76 0.073 5.1 0.013

Table 6.1 Kinetic parameters for the reduction of unnatural substrates by KR.203

Overall, it was clear that, within the range of compounds studied, act KR showed a

preference for bicyclic substrates. This lead to Korman, Tsai and co-workers to speculate

that the most likely substrate for KR was a hypothetical intermediate 82, where the first

cyclization (C7 to C-12 to form 83) and the second cyclization (C-6 to C-15 to form 82)

have already occurred (Scheme 6.4). The product of KR, a hypothetical intermediate 84,

would spontaneously cyclize to afford mutactin 20.

74 75 76 77 78


114

S-ACP

OOOO

O

O OO

O

O

OH

OH

HO

O

S-ACP

OOO

O

O OO

99

7

12 12

12

7

7

9

O O

O

O

O

12

79

OHOH

OS-ACP

O O

O

HO

O

12

79

OHOH

OS-ACP

6 6

66

6

15 15

151515

Scheme 6.4 Model proposed by the group of Tsai, in which the substrate for act KR is the bicyclic

intermediate 82.203

The model proposed by the group of Tsai has been challenged by Kalaitzis and Moore, who

heterologously expressed the actinorhodin minimal PKS genes, together with actIII (KR),

actVII (ARO) and actIV (CYC) (see Figure 1.6 in Chapter 1), in S. lividans.204 Two new

hexaketides BSM1 85 and BSM3 86 were characterized (Scheme 6.5) which arose from

partially completed polyketide structures, in which the ketoreduction had already taken

place.

Kalaitzis and Moore then proposed an alternative pathway for the biosynthesis of

polyketides, where ketoreduction occurs during the elongation of the polyketide chain

rather than after the assembly of the full length octaketide (Scheme 6.5).204 In this model,

the substrate for KR would be the pentaketide 87. After reduction at the C-3, the reduced

pentaketide 88 would be extended with a further malonate unit to the hexaketide 89. And

aldol condensation between C-3 and C-8 then would lead to 85 and 86.

The detection of BSM1 and BSM3 is unique in all Type II PKS studies in that it

describes the isolation of partially complete polyketides. This has never been reported in in

vitro assays. In fact, Moore left open the possibility that these hexaketides may be

degradation products resulting from the catabolism of S. lividans.

C-7 to C-12 cyclization

C-6 to C-15 cyclization

KR spontaneous

83

82 84 20

18


115

O

COOH

O

OH

O

COOH

O

S-ACP

O

O

O OO

3

1

65

CoA-S

O

OH

O

5 x

S-ACP

O

HO

O OO

3

1

65

O

HO

O OO

5

3

86

O

S-ACP

malonyl CoA

CoA

3

8

H2O

Scheme 6.5 Proposed biosynthesis of BSM1 and BSM3.204

Another example where ketoreduction was proposed to operate before the completion of

the polyketide chain was that of the enterocin (enc) 17 PKS. Hertweck, Moore and

collaborators expressed the enc ACP, KSα and KSβ in S. lividans in the presence of

benzoyl CoA. No polyketides could be detected.205 On the other hand, when KR was co-

expressed with ACP, KSα and KSβ, wailupemycins D-G 90-93 were produced (Scheme

6.6). It thus appeared that the corresponding enc minimal PKS would need the inclusion of

a fourth component, i.e. KR.

Indeed, polyketide production by enc ACP, KSα and KSβ was not recovered either by

complementation with act KR or with a mutant enc KR in which the catalytic serine active

site had been mutated to alanine.205 Thus it was clear, first, that KR was an essential

component of the minimal enc PKS and exerted a catalytic, rather than structural, role.

Second, that ketoreduction must occur during polyketide elongation. Although Hertweck,

Moore and co-workers proposed that the likely substrate for KR could be an hypothetical

pentaketide (Scheme 6.6), no evidence for this could be gathered –indeed, absolutely no

truncated polyketides could be observed. Therefore, if KR was not present, either the

85 86

89 88

87 KR

Act Min PKS

Act Min PKS


116

hypothetic polyketides resulting from 94 (or other intermediate) were not released from the

PKS, or they were simply catabolyzed by the host.

S-ACP

O

HO

Ph

O OO

CoA-S

O

OH

O

4 x

O

HO

Ph

O OO

O

S-CoA

O

S-ACP

OOO

O

OH

O

OH

HO

O

OO

OH

O

O

OH

R2

OH

HO

OH

O

O

OH

Ph

OH

HO

O

O

O

OH

Ph

OH

HO

OH

R1

Scheme 6.6 Biosynthesis of wailupemycin D-G by the enc minimal PKS. Wailupemycin D: R1 = OH, R2 =

Ph; Wailupemycin E: R1 = Ph, R2 = OH.205

In summary, no conclusive evidence has been found to date that unequivocally identifies

the substrate of act KR, the timing of the first and second cyclizations of the polyketide

chain, or how KR interacts with the rest of the components of the act PKS. This is

especially true in in vitro systems. Given the quantitative assay to measure reaction rates

which was developed in this work, we decided to adapt the method to measure mutactin

production by an extended act minimal PKS consisting of ACP, KSα/KSβ and KR. The

aim of this Chapter is therefore to gain insight into the catalytic properties of KR.

In the following sections, the structure and chemical mechanism of KR, as well as

seminal work by the Stanford group on the kinetics of the ketoreduction, are described.

Then, our results will be presented.

17

Enc ACP, KSα/KSβ and KR

Min enc PKS + 3 x malonyl CoA

Min enc PKS

Spontaneous

94

90, 91 D, E 92

F 93 G


117

6.1. Structure and chemical mechanism of KR

The first three-dimensional structure of a Type II PKS reductase, the actinorhodin KR, was

determined at 2.5 Å resolution by the Bristol group.32 The quaternary structure of act KR

was investigated by native PAGE. In the absence of NADPH, the enzyme appeared to exist

in different oligomeric forms (i.e. tetramer, dimer and monomer).206 Addition of NADPH

(or indeed NADP+) resulted in only one protein band corresponding to the expected

tetrameric molecular weigh.206 Crystallization of KR was therefore done in the presence of

NADP+ to ensure an active conformation of the enzyme.

KR belongs to the short-chain dehydrogenases/ reductases (SDR) protein family. These

are relatively small (250-350 residues) enzymes that show medium (15-30%) sequence

identity and display highly similar folding patterns, with a central β-sheet core flanked by α

helices.207 The Asn-Ser-Tyr-Lys catalytic tetrad of the SDR family is conserved in act KR

(Figure 6.1).

Figure 6.1 Structure of a monomer of act KR, showing the β-sheet core (blue) and the catalytic residues

N114, S144, Y157 and K161 (yellow). PDB file: 1W4Z.

Reduction of the ketone substrate is accompanied by a proton relay mechanism, which is

similar to that in other enzymes of the SDR family.202 Following hydride transfer from

NADPH to the substrate carbonyl, the alkoxide is stabilized by the oxyanion hole formed

by S144 and Y157 (Scheme 6.7), and the proton from the hydroxyl group of Y157 is


118

transferred to the carbonyl oxygen. A proton relay then occurs to account for the proton

extracted from Y157, sequentially involving the hydroxyl group of the NADPH ribose, the

amino group of K161 and four water molecules.

Y157

OH

O

OO

R NH

H

H

K161

NH H

R'

R''O

OH

S144

H

O

HOH

H

O

H

H

O

H

H

O

H

N114

Scheme 6.7 Proton relay mechanism in the act KR active site (blue arrows) showing NADPH (green), the

polyketide substrate (red) and the active site residues (black).202

6.2. Kinetics of extended Type II minimal PKS.

The Stanford group studied the production of polyketides when the act 9 and tcm 30

minimal PKS were complemented with auxiliary PKS enzymes such as act KR, gris ARO

and tcm ARO.11 The tcm minimal PKS assembles a decaketide from malonyl CoA, which

in the absence of further tailoring enzymes spontaneously cyclises to form mostly SEK15

95 and SEK15b 96, as well as RM80 97. Kinetic studies were performed using a

discontinuous, radioactive thin layer chromatography assay. The kcat value for total

decaketide production by the tcm minimal PKS was determined as 1.40 ± 0.25 min-1,11

substantially higher than kcat for the act minimal PKS (kcat = 0.31 ± 0.11 min-1).56

Addition of tcm ARO resulted in a relative increase in production of 97 with respect to

95 and 96 (Scheme 6.8). Furthermore, production of polyketides was enhanced by the

presence of ARO, and an increase in kcat to 2.55 ± 0.31 min-1 was measured by HPLC

analysis of products. This agreed with early work by Shen and Hutchinson which indicated

that the presence of further PKS enzymes also enhanced the rate of polyketide

production.188


119

O

OH

HO

O OH

OH

O OH

O

OH

HO

O OH

O

O OH

OH

HO

O

O

O OH

OH

O

10 x malonyl CoA

OH

CH3OHO

O

H3CO

O

OHOH

OO

OCH3

CH3

Scheme 6.8 Biosynthesis of decaketides by tcm PKS

In the actinorhodin PKS, addition of KR to minimal assays results in production of

mutactin 20 (Scheme 1.13). Further supplementation with actVII (an aromatase) affords

SEK34 21. When the corresponding aromatase from the griseusin PKS substituted actVII,

SEK34 21 was still produced.208

The Stanford group then assessed the production of mutactin and SEK34 by HPLC.

They found that inclusion of act KR in act minimal assays resulted in a decrease in the rate

of polyketide production (kcat = 0.11 ± 0.02 min-1),208 when compared with previous kinetic

data for the act minimal PKS (kcat = 0.31 ± 0.11 min-1)56

When gris ARO was added to KR-extended act minimal PKS assays, an increase in rate

was measured (kcat = 0.44 ± 0.04 min-1). Obviously this meant that the rate of ketoreduction

had been increased at least by a factor of four due to the presence of gris ARO, which they

attributed to KR-ARO channelling of polyketide intermediates.

6.3. Kinetic analysis of production of mutactin by an extended act minimal PKS

The actinorhodin his6-KR was expressed in E. coli and purified by nickel affinity

chromatography as described previously in the literature.32 6 mg pure KR per litre of

SEK15 95 SEK15b 96

RM80 97 30

Tcm min PKS

Tcm min PKS + ARO

Tcm PKS


120

culture were obtained. ESMS analysis of the enzyme showed the peak corresponding to KR

and a peak probably corresponding to the α-N-6-phosphogluconoylation of the His-tag

(Figure 6.2).209

Figure 6.2 Mass Spectrum of his6-KR (expected: 29291 Da, observed: 29299 Da) and α-N-6-

phosphogluconoylation of the His-tag (expected: 29467 Da, observed: 29477 Da).

Mutactin was purified from in vitro assays. 0.6 mg of mutactin were obtained by HPLC

separation of polyketides produced in the assays. This sample was further analyzed by

LC/MS (Figure 6.3). We used this standard for calibration of the HPLC system and for the

calculation of the extinction coefficient of mutactin at 293 nm (ε = 6,700 M-1 cm-1) for

kinetic purposes.

Production of mutactin by the act minimal PKS in the presence of KR was reconstituted

following the procedure of Zawada and Khosla.208 First, we studied the dependence of the

ratio mutactin vs. total polyketide production at a range of KR concentrations. Thus, holo-

ACP, KSα/KSβ and malonyl CoA were incubated with increasing concentrations of KR

and production of polyketides after 2 h assay was assessed by HPLC analysis, by

measuring the absorbance of the samples at 280 nm. In agreement with previous reports,208

we needed excess KR to favour the production of mutactin over SEK4/4b (Figure 6.4).


121

Figure 6.3 LC/MS analysis of mutactin. A. Chromatogram showing the mutactin peak at 26.40 min. B.

Individual mass chromatogram at 303 Da (expected mass of [M+H]+).

19 27 20

Figure 6.4 HPLC chromatograms at 280 nm showing the biosynthesis of SEK4 19, SEK4b 27 and mutactin

20 at fixed concentrations of malonyl CoA (1 mM), holo-ACP (50 µM) and KSα/KSβ (0.6 µM) and a range

of KR concentrations.

A.

B.

KR =0.25 µM

KR = 0.5 µM

KR = 1 µM

KR = 2 µM

KR = 3 µM

KR = 5 µM

KR = 10 µM

Time (min)

Time (min)


122

We then determined the percentage of the total polyketides that were reduced at every KR

concentration. The ready availability of pure samples of SEK4/4b (see Section 2.4.2.1) as

well as mutactin, allowed the calibration of the HPLC system. This facilitated the

quantification of the each polyketide by integration of the peak areas in the HPLC

chromatogram (read at 280 nm, Figure 6.4). This was done taking into account that SEK4,

SEK4b and mutactin possess different extinction coefficients at 280 nm. It turn out that

more than 80% of the total polyketides produced corresponded to mutactin when 10 µM KR

was used (Figure 6.5). This corresponded to a 20-fold excess of KR over KSα/KSβ (KR =

10 µM vs. 0.6 µM KSα/KSβ).

Figure 6.5 Effect of increasing concentrations of KR on the ratio mutactin: (SEK4 + SEK4b).

HPLC analysis of polyketides provided useful information about the reaction conditions

needed for mutactin production when the act minimal PKS was supplemented with KR.

However, it only provided a snapshot of the reaction: samples were analyzed by HPLC

after allowing the reaction for 2 h. As we intended to use the method developed in

preceding chapters to observe polyketide production directly by UV spectrophotometry in a

plate reader, it was imperative to check the ratio of products during the first minutes of the

experiments.

A time course study of polyketide production in the absence and presence of KR was

then done. This showed that mutactin was produced in excess of SEK4 and SEK4b over the


123

whole period of study (Figure 6.6). Interestingly, the ratio SEK4/SEK4b changed over

time; but the pattern was the same in both experiments.

19 27 19 27 20

Figure 6.6. HPLC chromatograms at 280 nm of the time course study of polyketide production (SEK4 19,

SEK4b 27 and mutactin 20) in the absence (A) and presence (B) of KR (10 µM).

6.3.1. Self-malonylation of ACP in the presence of KR

Two continuous assays are described in Chapter 2 to measure the rate of self-malonylation

of ACP. In principle, we could use the α-ketoglutarate dehydrogenase assay in the presence

of a mutant KSα/KSβ which is incapable of polyketide synthesis (Section 2.4.2.2) and

measure the rate of self-malonylation in the presence of both KSα/KSβ and KR. However,

KR needs NADPH (or at least NADP+), to be in the active, tetrameric conformation206 and

this complicated the use of α-ketoglutarate dehydrogenase, which itself uses NAD+ as a

substrate, as a coupling enzyme.

We then decided to use KSα/KSβ at high concentrations (3 µM) to couple the

production of polyketides to the formation of malonyl-ACP from holo-ACP and malonyl

CoA. First, we tested whether an increase in the concentration of KSα/KSβ affected the

ratio mutactin: SEK4/4b determined in Figure 6.7. Therefore the dependence of the ratio

mutactin vs. (SEK4+SEK4b) on the ratio KSα/KSβ: KR at a fixed concentration of KR was

B.

15 min

30 min

60 min

120 min

15 min

30 min

60 min

120 min

A.


124

assessed. Thus KR was fixed to 10 µM, and nine different concentrations of KSα/KSβ

between 0.3 and 3 µM were studied. After allowing reaction for 2 h, polyketides were

analyzed by HPLC. Integration of the peak areas and comparison with standards showed

that mutactin accounted for 81 ± 1 % of the total polyketides produced at all KSα/KSβ

concentrations (Figure 6.7).

This experiment suggested that the ratio KSα/KSβ: KR was unimportant to determine

the production of mutactin, when compared to SEK4 and SEK4b. However, the absolute

KR concentration was an essential variable to achieve an excess of mutactin, as shown in

Figure 6.4. Therefore the hypothesis that KR interacts with ACP, as proposed by the Bristol

and Tsai groups,32,202 might be valid.

Figure 6.7 HPLC chromatograms at 280 nm showing polyketide production (SEK4 19, SEK4b 27 and

mutactin 20) upon titration of KSα/KSβ in extended minimal PKS assays at fixed KR concentration (10 µM).

The study of the self-malonylation of act holo-ACP by the KSα/KSβ coupling system in

the presence of KR rendered kinetic parameters kcat = 2.41 ± 0.22 min-1 and KM = 226 ± 51

µM (Figure 6.8), which are in excellent agreement with those determined in the absence of

KR (Section 2.4.2.2).

19 27 20

0.3 µM

0.5 µM

0.75 µM

1 µM

1.25 µM

1.5 µM

2 µM

2.5 µM

3 µM


125

Figure 6.8 Determination of kinetic parameters for the self-malonylation of holo-ACP in the presence of KR

by the KSα/KSβ coupling system. A. Plot of reaction rate vs. substrate concentration and hyperbolic fit. B.

Hanes linear plot of the data.

6.3.2. Addition of MCAT to extended minimal PKS assays

Addition of MCAT to act minimal assays resulted in acceleration of ACP malonylation and

a change in the rate limiting step of the overall reaction (See Chapter 3). In the absence of

KR, this new rate limiting step was decarboxylation of malonyl-ACP by KSβ.

The kinetic study of the initiation step in the absence of KR produced the kinetic

parameters kcat = 20.6 min-1 and KM = 2.39 µM. In the presence of 10 µM KR identical

values were obtained (kcat = 20.6 ± 0.8 min-1 and KM = 2.32 ± 0.32 µM), Figure 6.9.

During the course of our studies, we did control reactions in the absence of NADPH.

Interestingly, we observed reduction in PKS activity (by ~ 50%) when ACP, MCAT,

KSα/KSβ and KR were incubated with malonyl CoA in assays where NAPDH was absent.

The quaternary structure of KR was recovered by inclusion of NADP+ in the assays;206

however, this not did not alter the result. Importantly, under these conditions SEK4 and

SEK4b were produced in the same ratio as in the absence of KR, after 2 h reaction.

A. B.


126

Figure 6.9 Determination of kinetic parameters in MCAT-supplemented minimal PKS assays in the presence

of KR. A. Plot of reaction rate vs. substrate concentration and hyperbolic fit. B. Hanes linear plot of the data.

6.3.3. Addition of acetyl-ACP to MCAT-supplemented, extended minimal PKS assays

Addition of acetyl-ACP (30 µM) to MCAT-supplemented act minimal assays in the

presence of 10 µM KR led to a 2.6-fold increase in rate, in agreement with our findings in

the absence of KR. Under these conditions, reactions rates depended linearly on the

concentration of KSα/KSβ.

6.4. Discussion

Numerous questions remain about the mechanism and specificity of PKS KR. For instance,

does KR interact with ACP, KSα/KSβ or both? How is the regiospecificity of the

ketoreduction at C-9 controlled? Does KR use as a substrate a full length octaketide? If this

is the case, is it the previously cyclised octaketide or the uncyclised intermediate? And

consequently, how does KR exert its influence on the regioespecificity of the first

cyclisation?

In Chapter 6 we have applied the methodology developed in preceding sections to study

the effect of KR on the kinetics of the act minimal PKS in vitro. The kinetic

characterization of the act minimal PKS in the presence of KR done here aimed to

contribute to the understanding of the reaction catalyzed by KR. The Stanford group

A. B.


127

observed a decrease in rate when act KR was added to act minimal PKS supplemented with

MCAT.11 In our hands, the rates of self-malonylation of holo-ACP, decarboxylation by

KSβ and chain elongation by KSα appeared to be unchanged upon addition of KR. Thus

the reduction of the polyketide catalyzed by KR must be faster than any of the preceding

steps and a full kinetic analysis of the ketoreduction catalyzed by KR was not possible by

the methods we have developed.

In the absence of KR, the act minimal PKS produces the unreduced octaketides SEK4 and

SEK4b. KR must interact with the minimal PKS, because the C10-C15 cyclisation route of

SEK4b is lost when KR is added to minimal PKS in vitro assays. In agreement with

previous reports by the Stanford group,11 we needed high concentrations of KR (10 µM) in

the assays in order to observe predominant production of the reduced polyketide mutactin.

The interaction between KR and KSα/KSβ cannot be limiting in this respect, because

the ratio KR: KSα/KSβ did not seem to alter the relative production of mutactin with

respect to SEK4 and SEK4b: the same 80% excess mutactin (over SEK4/4b) was observed

for a range of KR: KSα/KSβ ratios varying between 30 and 3.

Therefore the idea that KR interacts with ACP might be valid.32 KR could induce a

more rigid ACP-PP structure, thus restraining the degrees of freedom of the polyketide

chain in the tunnel between KSα and KSβ. This could favour the C7-C12 cyclisation

pattern of mutactin and prevent the ‘unnatural’ C10-C15 cyclisation of SEK4b.

The idea that KR interacts with ACP and not with KSα/KSβ is supported by the

structure of the mammalian FAS (see Section 1.3). In this type I FAS structure, the KS

domains form the body of the megasynthase whereas KR forms the ‘arms’, and their

corresponding active sites are 72 Å apart.85 ACP, on the other hand, is supposed to lie

between KS and KR, in the middle of each reaction chamber (see Figure 1.13 in Chapter 1).

Therefore, in the mammalian FAS it is reasonable to think that ACP is the link between KS

and KR domains, and direct interaction between KR and KS seems unlikely. In the act

PKS, ACP interacts with KSα/KSβ (Chapters 2, 3 and 4) and it also probably acts as the

link between KSα/KSβ and KR.

An interesting observation was the reduction in PKS activity when ACP, MCAT, KSα/KSβ

and KR were incubated with malonyl CoA in the absence of NADPH (but in the presence

of NADP+). Under these conditions, SEK4 and SEK4b were produced in the same ratio as


128

in the absence of KR, but at a lower rate. Assuming that the quaternary structure of KR is

conserved in the presence of NADP+, as suggested by the Bristol group,206 this result would

mean that an ‘inactive’ KR partially impairs polyketide production by the act PKS.

This finding is reminiscent of the Hertweck, Moore et al work when they could not

reconstitute the enc minimal PKS in vivo, either in the absence of enc KR, or indeed in the

presence of an inactive enc KR (see introduction to Chapter 6).205 Therefore it appeared

that ketoreduction in the enc PKS must occur in order for enterocin-wailupemycins

polyketides to be produced.

Similarly, we could argue that ketoreduction must occur in order for the C-7 to C-12

cyclisation to be predominant in the act PKS. This would imply that cyclisation of the

polyketide chain occurs after the KR step. Evidence for this comes from our finding that, if

a structurally competent but catalytically inactive act KR (i.e. KR without NADPH) is

present in act in vitro assays, SEK4b is still produced. Therefore KR must perform a

catalytic role in the cyclisation of the polyketide chain, and this is irrespective of any

structural constrain KR may impose on the ACP-PP-polyketide chain structure, as

speculated above.

This hypothesis is in agreement with recent findings from Ma, Zhang and collaborators,

who found that act KR was able to reduce a polyketide produced by a fungal PKS.210 PSK4

from Gibberella fujikoroi synthesizes the nonaketide 97 from malonyl CoA. When act KR

was added to the assay, mutactin 20 was also produced. This is striking because 97 does not

show a C-7 to C-12 cyclisation. However, the action of act KR induced production of

mutactin, a C-7 to C-12 cyclised polyketide. This result strongly suggests that

ketoreduction by act KR occurs prior to cyclisation of the polyketide chain.

O

O OH OH

OHHO

2

79

1012

13

17

97


129

7. Conclusions

The act PKS has proven to be a very useful model for understanding polyketide

biosynthesis. For example, the act PKS is a good model for the iterative Type I PKS found

in fungi, because the act PKS is also iterative; that is, the single set of proteins acts several

times to produce a single final product (i.e. actinorhodin). Also, the individual reactions

catalyzed by the act PKS are the same as those catalyzed by the chain extension domains of

single modules of bacterial Type I modular polyketide synthases. Finally, the act PKS is

also a model for other Type II polyketide synthases in bacteria. Thus, understanding of the

act PKS serves to underpin advances in understanding the main classes of polyketide

synthase proteins in bacteria and fungi.

The act PKS has been studied for more than 20 years, from the identification and

cloning of the corresponding gene cluster to the study of the enzymology of purified

proteins. Within the act PKS, there are three proteins essential for production of

polyketides. ACP, KSα and KSβ thus form the act minimal PKS. During the last decade,

production of polyketides by the act minimal PKS has been studied mainly by two groups,

i.e. the Stanford and the Bristol groups. In 2006, the Bristol group published a model for

the production of SEK4 and SEK4b by the act minimal PKS in vitro (Scheme 1.13).60 We

accepted this model as the starting point.

In this work, we have developed continuous methods to measure the rate of succeeding

steps in the act minimal PKS. The chemical reactions catalyzed by the act minimal PKS

were grouped into loading, initiation, chain extension, and cyclisation and release steps

(Scheme 1.18). The loading step (i.e. self-malonylation of holo-ACP) was the rate limiting

step in act minimal PKS assays, as suggested by early work in the Bristol group.8

Experimentally, loading rates could be studied under conditions where KSα/KSβ was

added in high concentrations (Chapter 2).

In order to accelerate the rate of the loading step, MCAT was added to our assays.

MCAT provided rapid formation of malonyl-ACP, so that loading was no longer rate

limiting. Under these conditions, it appeared that initiation of polyketide synthesis by

decarboxylation of malonyl-ACP by KSβ was slower than chain elongation catalyzed by

KSα (Chapter 3). Addition of excess starter unit in the form of acetyl-ACP allowed the


130

measurement of the slowest reaction catalyzed by KSα. It appeared that cyclisation and

release of the polyketide chain to form SEK4 and SEK4b occurred at an even higher rate

(Chapter 4).

Therefore a methodology was developed to study the kinetics of each of the enzymes

that constitute the act minimal PKS, as well as KR (Chapter 6). During the course of our

studies, we have obtained evidence for protein-protein interactions among the components

of the act minimal PKS, which have contributed to understanding the molecular

architecture of the complex.

7.1. Self-malonylation of holo-ACP is aided by KSα/KSβ

The study of protein-protein interactions between ACP and KSα/KSβ was prompted by the

discovery that ACP and KSα/KSβ were closely associated during the loading step. The rate

of self-malonylation increased five-fold when holo-ACP was in the presence of KSα/KSβ

(Section 2.4.2). We attempted to gain insights into the ACP: KSα/KSβ association by the

study of the self-malonylation of act mutant acyl carrier proteins. Thus we chose two act

mutants (E47A and E53A) previously shown to self-malonylate with the same rate as the

reference act ACP by a mass spectrometry assay. E47 and E53 had been proposed to have a

role in ACP: KSα/KSβ interactions.120

In our hands, however, these mutant acyl carrier proteins were deficient in self-

malonylation when studied in isolation, and thus we lacked of an appropriate control to

study ACP: KSα/KSβ interactions during the loading step (Section 2.5.1). Because of this,

the nature of this association remains unclear. It is tempting to speculate that holo-ACP

adopts a different, perhaps more rigid, conformation in the presence of KSα/KSβ, leading

to enhanced catalytic activity. But structural studies performed within the Bristol group

have shown that holo-ACP in isolation is itself a rigid protein, and the inherent flexibility of

other FAS and PKS acyl carrier proteins has not been observed with act ACP.193

The ACP: KSα/KSβ interaction during the loading step was highly specific: the rates of

self-malonylation of gris ACP and dps ACP did not undergo any change in the presence of

act KSα/KSβ. Future work should investigate this further by the generation of more mutant

acyl carrier proteins and kinetic characterization of their self-malonylation rates. NMR


131

titrations of KSα/KSβ to WT and mutant holo-ACP solutions could help proving the

hypothesis of the conformational change. Eventually, residues on the surface of KSα/KSβ

could also be mutated.

A key question raised by our in vitro results is their relevance to the situation in vivo. It is

clear that the minimal act PKS consisting of ACP and KSα/KSβ is competent to produce

octaketides over a wide range of protein and malonyl CoA concentrations (Chapter 2), but

does it do this efficiently? In the absence of MCAT the rate limiting step is the self-

malonylation of ACP. The rate of this reaction depends on the concentrations of malonyl

CoA and holo-ACP and increases linearly with holo-ACP concentration (Figure 2.12). We

have also studied how the overall rate of octaketide production depends on holo-ACP

concentration (Figure 3.2). At concentrations above about 80 µM holo-ACP and saturating

concentrations of malonyl CoA, the rate of self-malonylation is sufficient to match or

outpace the requirement for malonyl-ACP by KSα/KSβ. At lower holo-ACP

concentrations, MCAT accelerates malonyl transfer, and for concentrations of holo-ACP

below 80 µM, the presence of MCAT speeds up the overall reaction.

The determination of kinetic parameters in the absence and presence of MCAT allows

the prediction on how the behaviour of the MCAT-catalyzed malonylation of holo-ACP

and the self-malonylation of holo-ACP in the presence of KSα/KSβ vary with the

concentration of ACP, MCAT, and malonyl CoA (Figure 7.1). The kinetic parameters for

act holo-ACP as a substrate for MCAT have not been measured because of the self-

malonylation reaction, but by comparison with S. coelicolor FAS ACP it is reasonable to

estimate a KM of 60 µM and kcat of 450 s-1.46 It is clear that under conditions such as high

MCAT concentration (>10 nM), low ACP concentration (<80 µM), and low malonyl CoA

concentration (<100 µM), the MCAT catalyzed reaction would be expected to dominate

(Figure 7.1). However it is also possible for the self-malonylation reaction to dominate, for

example at high ACP (>80 µM) and malonyl CoA concentrations (>400 µM) and low

MCAT concentrations (<5 nM).

The in vivo concentrations of malonyl CoA and act holo-ACP have never been

measured in S. coelicolor, but Rock and co-workers have estimated the concentration of E.

coli FAS ACP as between 17 and 130 µM in vivo, and malonyl CoA concentrations have

been estimated between 110 and 1000 µM.166 Few in vivo experiments have been performed


132

to correlate individual protein concentrations with the rate of polyketide biosynthesis.

However, Khosla, Hopwood, and co-workers showed in an elegant series of experiments

that likely increases of in vivo ACP concentrations correlate well with the yield of

polyketides.211 This is in agreement with similar observations of Hutchinson and co-

workers using the tetracenomycin PKS.212 The fact that ACP appears to be limiting in vivo

correlates well with observations reported here that ACP concentration is limiting in vitro

up to around 80 µM (Figure 3.2).

MCAT concentrations are also unknown in vivo. Studies of the S. coelicolor MCAT in

vitro have used concentrations between 0.5 nM and 100 nM.46,56 If in vivo MCAT

concentrations were at the lower end of this range, then our results indicate that self-

malonylation would be dominant and malonylation of the ACP would be the rate limiting

step in vivo. If, however, MCAT concentrations were at the upper end of the range, then

MCAT-catalyzed malonylation would dominate, and provision of acetyl ACP for use as a

starter unit would be rate limiting for the PKS.

Figure 7.1 Comparison of the behavior of MCAT-catalyzed malonylation of holo-ACP vs self-acylation of

act PKS holo-ACP. Linear plots show how the rate of self-malonylation of holo-ACP in the presence of

KSα/KSβ varies with ACP concentration at two concentrations of malonyl CoA (rate data from Figure 2.6).

Curves show the effect of saturation catalysis by MCAT at differing MCAT concentrations (for simulated

MCAT with KM of 60 µM for ACP and kcat of 450 s-1 at saturating malonyl CoA). Grey horizontal line shows

demand for malonyl ACP by KSα/KSβ at 0.75 µM.

1000 µM mal-CoA

100 µM mal-CoA

10 nM MCAT

5 nM MCAT


133

The demand for malonyl ACP must also be considered as well as the supply. Our results

show that in the absence of excess acetyl ACP, octaketides are produced by KSα/KSβ at

20.6 min-1 which corresponds to the use of malonyl ACP at 165 min-1. For a KSα/KSβ

concentration of 0.75 µM (typical for in vitro studies), this corresponds to a malonylation

rate of 123 µM·min-1; this is shown as the grey line in Figure 7.1. It is clear that this could

be supplied by ACP at approximately 70 µM and malonyl CoA at 1000 µM in the absence

of MCAT or by 55 µM ACP and saturating malonyl CoA in the presence of 10 nM MCAT.

Thus, the concentrations of all catalytic and substrate participants are required in order to

determine which pathway dominates in vivo. While the results reported here show which

factors are important and give limits for each process, further experiments will be required

to determine these concentrations in vivo during actinorhodin biosynthesis.

7.2. Quaternary structure of KSα /KSβ and ACP: KSα /KSβ complexes

In order to study the quaternary structure of KSα/KSβ, we assayed mutant KSα/KSβ using

the methods developed to study the kinetics of KSα and KSβ separately. For example, the

decarboxylation activity of a CA KSα/KSβ was assessed (Section 5.1.1). As expected, this

mutant was deficient in decarboxylation of malonyl-ACP, due to the mutation of the active

site in KSβ. Addition of a mutant KSα/KSβ incapable of polyketide synthesis (i.e. AQ

KSα/KSβ) to CA KSα/KSβ assays resulted in an increase of the rate of SEK4/4b

production under saturating concentrations of acetyl-ACP (Section 5.3) This meant that the

presence of AQ enhanced the production of polyketides by CA KSα/KSβ by a different

means than production of acetyl-ACP. This result could be explained if the catalytic

KSα/KSβ species were the dimer of heterodimers, and then addition of {AQ|AQ} to

{CA|CA} resulted in some {CA|CQ} complex being formed. This {CA|CQ} complex

would then produce polyketides more efficiently than {CA|CA}.

We speculate that decarboxylation of malonyl-ACP by KSβ is accompanied by a

conformational change of KSα/KSβ (see discussion to Chapter 5). This could account for

the observed increase in rate caused by introduction of an active KSβ in the tetramer. This

conformational change is not prompted by substrate binding onto KSβ (see discussion to

Chapter 5), and may happen as a result of malonyl-ACP decarboxylation. This makes sense


134

because the following reactions are catalyzed by KSα, which is now in an active

conformation. Further evidence to support the hypothesis of the conformational change was

provided by the experiments in which we added excess acetyl-ACP to CA KSα/KSβ

assays. In these assays, an increase in rate of only 3-fold (kcat changed from 2.2 to 6.4 min-

1) was observed, instead of the expected 30-fold to reach WT levels. This suggested that

CA KSα/KSβ was deficient in more than just the initial decarboxylation of malonyl-ACP

(see discussion to Chapter 4).

We propose here that the crystal structure of KSα/KSβ published by the Stanford group

in 2004,31 corresponds to the ‘closed’ (inactive) form of KSβ and ‘open’ (active) form of

KSα; i.e. it represents a complex ready for extension, rather than for initiation. This could

explain why the active site of KSβ appeared to be buried. Indeed, electron density of

intermediates bound to KSα was identified.31

The stoichiometry of ACP: KSα/KSβ interactions was addressed by the study of the

inhibition of polyketide production by apo-ACP (Section 5.2). Whereas apo-ACP did not

appear to inhibit holo-ACP self-malonylation or indeed MCAT, it was a mixed inhibitor of

KSα/KSβ. Thus it seemed that KSα/KSβ possessed two different binding sites for ACP. In

principle, this could mean either that ACP interacted with each KSα/KSβ dimer (i.e. a 1:1

ratio between ACP and KSα/KSβ); or that KSα and KSβ within each dimer had different

binding sites for ACP (i.e. a 2:1 ratio between ACP and KSα/KSβ). In the first scenario, we

would expect very similar values for competitive and uncompetitive inhibition constants.

However, Kic was 50 µM whereas Kiu was 137 µM. These data are consistent with a model

in which each ketosynthase subunit in the KSα/KSβ tetramer has the ability of binding a

molecule of ACP (Figure 7.2).

KS! KS"

KS! KS"

ACP

ACP

ACP

ACP

Figure 7.2 Model for ACP: KSα/KSβ that contemplates four molecules of ACP per tetramer KSα/KSβ.


135

The interaction of ACP with KSβ was then studied. In agreement with previous models,120

we identified a couple of negatively charged residues within helix II of ACP as key players

in ACP: KSβ interations. Indeed, mutation of E47 or E53 to alanine residues resulted in a

3-4 fold increase in KM with unchanged kcat (Section 3.2) Moreover, the assays revealed

that KSβ binds malonyl-ACP (KM = 2.4 µM) selectively over apo-ACP, despite the little

structural difference between both species.

The interaction of ACP and KSα could not be fully studied due to the uncertainty in the

determination of KM for malonyl-ACP (Section 4.2). However, the fact that the introduction

of a bulky valine residue in position 47 of ACP had the same disadvantageous kinetic effect

in KSα and KSβ suggested that the binding site for ACP in both proteins is very similar.

Finally, the interaction of KR with ACP: KSα/KSβ complexes was studied (Section 6.3).

The ratio mutactin: SEK4/4b depended strongly on the absolute concentration of KR, but

not on the ratio of KR: KSα/KSβ, meaning that KR may not interact with KSα/KSβ.

Consisting with this, the self-malonylation rate of ACP in the presence of KSα/KSβ did not

change when KR was added: KR did not have an effect on the ACP: KSα/KSβ interactions

that lead to enhanced self-malonylation activity of holo-ACP. These results are consistent

with previous models described in the literature,32,202 which suggest that KR interacts with

ACP rather than with KSα/KSβ. This also makes sense when the structure of the

mammalian FAS (best current model for the act PKS) is taken into account (see discussion

to Chapter 6): ACP might be the link between the ketosynthase and the ketoreductase

activities in both models.

7.3. Model for an extended act minimal PKS

Based on the currently accepted model for the act minimal PKS,60 we propose here simple

refinements that can account for our observations (Scheme 7.1). Self-malonylation of holo-

ACP seems to be more efficient in association with KSα/KSβ (kcat ~ 2.3 min-1), rather than

in solution (kcat ~ 0.5 min-1). Therefore the loading step is likely to occur when ACP is

bound to the KSα/KSβ complex. Consequently, the ACP-ACP transfer activity which

occurs between acyl carrier proteins,60 might not be of biological importance.


136

The initiation step is catalyzed by KSβ, which generates acetyl-ACP. This acetyl-ACP most

likely is released into solution. Simultaneously, there is a conformational change in the

KSα/KSβ tetramer which renders a KSα that is now in an ‘open’ conformation. KSα then

binds acetyl-ACP and is primed with the acetyl starter unit. Extension of the polyketide

chain then occurs.

The reduction by KR probably occurs before the first cyclisation, because when KR

was added to minimal PKS assays in the absence of NADPH, SEK4b was still produced.

This means that the ‘correct’ C7-C12 first cyclisation is probably only favoured after

reduction of C-9 by KR (Section 6.3).

HS

ACP

KS! KS"

CO2

KS! KS"

ACP

S

O O

OH

ACP

S

O

ACP

S

O

ACP

S

O

mal CoA

CoA

SHO

OO

ACP

KR

CoA

Mal CoA

ACP

HO

O

O

O

OH

O O O

S

S

O

O

O

O

O

n

KS! KS"

KS! KS"

KS! KS"

KS! KS"

KS! KS"

KS! KS"

KS! KS"

KS! KS"

KS! KS"

KS! KS"

KS! KS"

KS! KS"

Scheme 7.1. Model for an extended act minimal PKS. Progressing clockwise from bottom left, self-

malonylation of ACP in association with KSα/KSβ; initiation reaction and dissociation of acetyl-ACP into

solution; priming of KSα with acetyl; extension of the polyketide chain by Claisen-type condensations;

reduction of polyketide by KR; and cyclisation and release. For clarity, only one reaction chamber is shown.

The timing of the reduction has not been elucidated in this work and n can vary from 1 to 4.

Loading

Initiation

KSα encounters acetyl-ACP in solution

Priming

Extension

Mutactin 20


137

Our kinetic data demonstrate that the loading step is the rate limiting step in the act minimal

PKS in vitro (kcat ~ 2.3 min-1). In the presence of excess malonyl-ACP, the initiation step

catalyzed by KSβ becomes rate-limiting (kcat ~ 20 min-1). If acetyl-ACP is present in

excess, the rate increases 3-fold more (kcat ~ 60 min-1), which most likely represents the rate

of the extension steps catalyzed KSα. We were unable to measure rates for the cyclization

and release steps, but these are likely higher still, because no accumulation of intermediates

attached to ACP or released into solution could be detected. The observed reaction rate in

the presence of excess malonyl and acetyl-ACP remained unchanged upon addition of KR,

which also indicates that cyclisation and release are not rate limiting, because the addition

of KR necessarily affects the cyclisation of the polyketide chain.

The results presented here show that individual catalytic steps of the act PKS can be

dissected. The act minimal PKS appears to be optimized for the conversion of malonyl

CoA to octaketides, because individual rate constants measured for the individual catalytic

steps show an increase in value for each succeeding step.

The biosynthesis of malonyl CoA from acetyl CoA (which is obtained from pyruvate in

the citric acid cycle) is catalyzed by an enzyme known as acetyl CoA carboxylase. This

reaction requires energy in the form of ATP.101 Therefore it makes sense that the

malonylation of ACP is the rate limiting step in the act PKS, ensuring that malonyl-ACP is

produced at the rate it is consumed by the PKS. This guarantees no accumulation of

malonyl-ACP in the cell, and no waste of malonyl CoA or ATP.

In vitro, the loading step can be accelerated by addition of MCAT. In these conditions,

the initiation step (i.e. decarboxylation of malonyl to acetyl-ACP) becomes rate limiting.

Because the cell needs energy to make malonyl-ACP, it also makes metabolic sense that

acetyl-ACP is not accumulated, and rapidly used by KSα to form polyketides. In vitro, the

elongation step catalyzed by KSα is ca. three times as fast as the initiation step catalyzed

by KSβ. Successive reactions catalyzed by the act PKS might be even faster. For instance,

the ketoreduction catalyzed by KR appeared to be faster than the elongation step (Section

6.3.3). Other methods than those developed in this work need be introduced to enable the

characterization of the kinetics of KR and further tailoring enzymes in the act PKS.


138

8. Experimental procedures

8.1. Bacterial strains and plasmids

8.1.1. Escherichia coli

RapidTrans™ TAM1 competent cells (Active Motif) were used for high-throughput

bacterial transformation and isolation of plasmids. BL21(DE3)pLysS competent cells

(Promega) were used for high efficiency protein expression of the desired genes (Table

8.1).

Plasmid Product, Resistance Reference / Supplier

pRJC001 Act C17S holo-ACP, Amr Dr. Russell J. Cox

pIJ2367 Act C17S apo-ACP, Amr Dr. Russell. J. Cox

pCJAII136/1 Act E47A C17S holo-ACP, Amr Dr. Christopher J. Arthur pCJAII124/1 Act E47V C17S holo-ACP, Amr Dr. Christopher J. Arthur pCJAII81/1 Act E53A C17S holo-ACP, Amr Dr. Christopher J. Arthur pRJC002 Gris WT holo-ACP, Amr Dr. Russell J. Cox

pPBIV165 Act R72A C17S holo-ACP Pedro Beltran

pPWDpsG WT holo-DpsG, Spmr Dr. Pakorn Wattana-Amon

pLHSIII67A S. coelicolor ACPS, Amr Dr. Leah Smith

pIJ2371 S. coelicolor MCAT, Amr Dr. Russell J. Cox

Table 8.1 Plasmids transformed into E. coli

8.1.2. Streptomyces coelicolor

S. coelicolor A3(2) CH999 lacks the entire set of genes of the actinorhodin cluster. The

group of Cambridge carried out the transformation of this strain with the plasmid carrying

wild type of mutant KSα/KSβ and provided mycelium for further growth and purification

of protein in our laboratory.


139

Plasmid Product, Resistance Reference / Supplier pCB84 his6-act KSα:KSβ, Kmr Dr. Christian Bisang

pCB91 his6-act KSα:[Q161A]KSβ, Kmr Dr. Christian Bisang

pCB56 his6-act [C169A]KSα:KSβ, Kmr Dr. Christian Bisang

pSEK4 act KSα:KSβ, act ACP, act

CYC, actIV, Ampr

24

Table 8.2 S. coelicolor A3(2) CH999 strains used in this work.

8.2. DNA techniques

8.2.1. Site directed mutagenesis

Site-directed mutagenesis of ACP was performed using the Stratagene QuikChange™

Mutagenesis Kit. Mutagenesis reactions were performed according to the manufacturer’s

instructions and contained 0.2 mM dNTP mixture, 50 ng dsDNA template (pRJC001), 4%

v/v dimethylsulphoxyde (DMSO), 125 ng forward primer, 125 ng reverse primer (Table

8.3), 1 unit KOD Hot Start polymerase enzyme, 1 mM MgSO4 and 10% v/v polymerase

reaction buffer in 50 µl total volume (made up with dH2O). The reaction was temperature

cycled using a MJ Research PTC-100 programmable temperature controller. Samples were

heated at 95°C for 30 seconds, followed by 18 cycles of 95 ˚C for 30 seconds, 60°C for 1

min and 72 °C for 2.5 min. 9 units of DpnI restriction enzyme were added once the PCR

reaction had completed and the template digested for 4 hours. The PCR product was

transformed into E. coli TAM1 competent cells for plasmid isolation.

Forward primer GTC GAC ACG CCG GCT GAG CTG CCT GAC

Reverse primer GTC GAG CAG CTC AGC CGG CGT GTC GAC

Table 8.3 Primers for site-directed mutagenesis of R72 ACP. Codons containing the mutation R72A are

underlined.


140

8.2.2. Plasmid DNA isolation and characterization

Plasmid DNA (1-10µg) was isolated from overnight cultures of E. coli TAM1 competent

cells (LB medium, 4 ml) using GeneElute™ Plasmid Miniprep Kit (Sigma), according to

the manufacturer’s instructions.

DNA agarose electrophoresis was performed using a Sigma horizontal gel

electrophoresis tank at an applied voltage of 200V (100A) connected to a Consort

microcomputer power supply unit. Staining of DNA fragments by soaking the agarose gel

in ethidium bromide (1 mg/l) allowed for the visualization of DNA bands under ultraviolet

light. Size and concentration of DNA plasmids were estimated by comparison with

standards (Hyperladder I, Sigma). pET15b plasmid based DNA samples of concentration

50-100 ng/µl were sequenced by Lark Technologies using T7 promotor.

8.3. Bacterial culture tecniques

Media ingredients were purchased from Sigma, Difco or Fisher, unless otherwise stated.

Distilled water was used for all media and solutions. All solid and liquid media was

sterilised prior to use (Astell Cientific ASA270, 121 ˚C, 15 psi, 15 min). All pH

determinations of media and buffers were undertaken using a Gelplas double junction

combination electrode (BDH) attached to a model 292 pH-meter (Pye Unicam).

8.3.1. Liquid media

8.3.1.1. SOC medium

SOC medium was used for the post-transformation recovery of E. coli. Tryptone (2 g),

yeast extract (1.5 g) and NaCl (0.5 g) were dissolved in water, then KCl (1 ml from 250

mM stock) was added and the volume made up to 90 ml. The pH was adjusted to 7.0 with 6

M NaOH and the total volume made up to 100 ml. After sterilisation, 2 ml of sterile 1 M

glucose and 1 ml sterile 1 mg/ml MgCl2 were added.


141

8.3.1.2. Luria-Bertani (LB) medium

LB medium was used for growth of E. coli either for plasmid isolation (3-5 ml) or protein

expression (1-4 L). Tryptone (10 g), yeast extract (5g) and NaCl (10 g) were dissolved in

900 ml water, and the pH adjusted to 7.5 with 6 M NaOH. The total volume was then made

up to 1 L. 200 ml were dispensed into 500 ml flasks which were covered with sponge

bungs and aluminium foil, and autoclaved.

8.3.1.3. Super-YEME (SY) medium

SY medium was used for growth of S. coelicolor. Tryptone (5 g), yeast extract (3 g), malt

extract (3 g), MgCl2 (1.1 g), glycine (5 g), L-proline (75 mg), L-arginine (75 mg), L-

cysteine (75 mg), L-histidine (100 mg) and uracil (15 mg) were dissolved in 800 ml water

(Solution A). Separately, glucose (10 g) and sucrose (340 g) were dissolved in 200 ml

water (Solution B). All solutions were autoclaved. Before use, 20 ml of Solution B were

dispensed in 10 x 500 ml sterilised flasks containing coiled springs and 80 ml of Solution

A.

8.3.2. Solid media

Solid media have been used to allow for selection of a single bacterial colony under

antibiotic selection markers. Antibiotic (100 µg/ml carbenicillin and kanamycin or 50

µg/ml spectinomycin and thiosthrepton according to the resistance gene in the

corresponding plasmid) was added after first melting and then cooling the medium to

approximately 60°C. The media was then poured (20 ml) into Petri dishes and allowed to

solidify.


142

8.3.2.1. Luria-Bertani agar (LB agar) growth medium

LB agar was the solid media used to plate out E. coli after transformation. 1.5% agar was

added to LB liquid medium and autoclaved. Plates were kept at 37 ˚C for 30 min prior to

plating out bacteria.

8.3.2.2. Mannitol Soya Flour Medium (SFM)

SFM was used to select a single colony of S. coelicolor for protein expression purposes. 2 g

agar, 2 g mannitol and 2 g soya flour were dissolved in 100 ml tap water and autoclaved

twice.

8.3.2.3. R5 Medium

R5 Medium was used to grow S. coelicolor A3(2) CH999/pSEK4 for isolation of SEK4 and

SEK4b. A solution was made up with 103 g/l sucrose, 0.25 g/l potassium sulphate, 10.12

g/l magnesium chloride hexahydrate, 10 g/l glucose, 0.1 g/l Difco casaminoacids, 3 ml

trace element solution (40 mg ZnCl2, 200 mg FeCl3.6H2O, 10 mg CuCl2.2H2O, 10 mg

MnCl2.4H2O, 10 mg Na2B4O7.10H2O and 10 mg (NH4)6Mo7O24.4H2O in 1000 ml distilled

water), 5 g/l yeast extract and 5.73 g/l TES buffer. A total volume of 1.5 L was made. 100

ml of this solution was poured into 250 ml flasks each containing 2.2 g agar and

autoclaved. At time of use, the medium was re-melted and the following ingredients were

added: 1 ml KH2PO4 (0.5%), 0.4 ml CaCl2.2H2O (5 M), 1.5 ml L-proline (20%), 0.7 ml

NaOH (1 M) and 100 µl thiosthrepton.


143

8.4. Extraction and purification of SEK4 and SEK4b

8.4.1. Growth of bacteria and extraction of polyketides

Growth of bacteria and extraction and purification of aromatic polyketides was executed

following published methods.8,24 Frozen S. coelicolor A3(2) CH999/pSEK4 mycelium was

grown up in 100 ml SY medium for 4 days at 30 ˚C, 220 rpm under thiostrepton selection

and then transferred to 60 plates (1.5 L R5 medium with 50 µg/ml thiostrepton). Plates

were incubated at 28 ˚C for 9 days and then diced and extracted in 3 litres of ethyl acetate

and methanol (4:1 EtOAc:MeOH) with 1% acetic acid. The solvent was dried (MgSO4) and

evaporated. The solid, dark brown residue was re-suspended in EtOAc, the solution then

filtered and evaporated. The weight of the remaining products was 3 g. This was re-

suspended in EtOAc and loaded into a Silica 60 column, which was flushed with 200 ml of

a EtOAc:Hexane:Acetic acid (80:17:3) mixture and then with 750 ml of acetonitrile. Thin

layer chromatography (70% EtOAc, 30% petroleum ether, 1% acetic acid) was used to

follow the aromatic rich fractions (by UV absorbance). The MeCN fractions showing

aromatic absorbance with an Rf of 0.35 were gathered together, the solvent evaporated and

the products re-suspended in EtOAc and applied to a second Silica column, run under

EtOAc:Hexane:Acetic acid (80:17:3). The same solvent was used to perform the TLC

analysis, and the aromatic compounds detected with an Rf of 0.61.

8.4.2. Liquid Chromatography/ Mass Spectrophotometry (LC/MS) analysis

LC/MS was used throughout the purification protocol to chase SEK4 and SEK4b. LC was

run in a Luna 5u C8(2) 150 x 2 mm column (Phenomenex) with water and acetonitrile

(both + 0.05% TFA) as mobile phase at a rate of 0.5 ml/min. A gradient from 5 to 95 %

acetonitrile was executed over 20 min (Waters 600 HPLC Pump). The UV absorbance of

the sample was monitored in a Waters 996 Photodiode Array Detector. Mass spectrometry

data was subsequently acquired by a Platform Micromass Electrospray Mass

Spectrophotometry facility (positive ion mode).


144

8.4.3. Preparative HPLC for purification of SEK4 and SEK4b

The separation of SEK4 and SEK4b was achieved by HPLC (Beckman). An Anachem C18

column was used with flow rate of 4 ml min-1. The mobile phase was water/acetonitrile (30

to 50% MeCN over 10 min) controlled by a Programmable Solvent Module 126. Detection

of polyketides by UV absorbance (Diode Array Detector Module 168) allowed the

collection of SEK4 and SEK4b (Autosampler System Gold 157).

8.5. Growth of bacteria and purification of proteins

8.5.1. General methods and equipment

8.5.1.1. Centrifugation

Large volumes were centrifuged either in the SLA-1500, SLA-3000 or GS-3 rotors in a

RC5C centrifuge (Sorvall instruments) at maximum speeds of 14,500, 11,000 and 9,000

rpm respectively. Medium volumes were centrifuged in a SS-34 rotor at 18,000 rpm. Small

volumes were centrifuge in eppendorf-centrifuges at 13,000 rpm (MSE microcentaur

centrifuges).

8.5.1.2. Shakers

Shakers were from New Brunswick Scientific, and allowed the control of temperature and

loads of 20 x 500 ml flasks.

8.5.1.3. UV Spectrophotometer

The UV Spectrophotometer was an Ultrospec III from Pharmacia. 1.5 ml plastic cuvettes

were purchased from Sarsdted.


145

8.5.1.4. Plate reader

Supplied by Molecular Devices, a SpectraMax 190 Plate Reader was used to measure the

absorbance of small volumes (up to 200 µl) in Corning UV-Transparent 96 wells

microplates.

8.5.1.5. Sonication

Cell-free extracts were obtained after lysing the cells by sonication (MSE Soniprep 150).

Sonication of E.coli was executed in 5 short bursts of 30 seconds with 1.5 min ice-cooling

in between. S. coelicolor was sonicated 10 times for 30 seconds.

8.5.1.6. Buffers

All buffers were made up with distilled buffer. Buffers for FPLC were filtered (0.45 µm) at

reduced pressure and degassed. Buffers for HPLC were filtered (0.2 µm) and degassed.

Buffers for proteins assays were filtered-sterilised (0.2 µm).

8.5.1.7. NTA-His-Bind nickel affinity chromatography

Nickel chromatography chromatography (NTA His-Bind resin -Novagen) was executed

following the manufacturer’s instructions. The column, which was stored in 50% ethanol,

was charged with 50 mM NiSO4 and equilibrated in binding buffer. The sample (typically

100 ml) was then loaded and the column subsequently washed with binding and washing

buffers. The protein of interest (ACPS, MCAT or KSα/KSβ) was collected in elution

buffer.


146

Addition Binding Buffer Washing Buffer Elution Buffer Stripping Buffer Phosphate Buffer 100 mM 100 mM 100 mM 20 mM Tris-Cl NaCl 0.5 M 0.5 M 0.5 M 0.5 M Imidazole 5 mM 60 mM 1 M 100 mM EDTA Glycerol 10% 10% 10% - pH 7.5 7.5 7.5 7.9

Table 8.4 Buffers used for Nickel affinity chromatography

8.5.1.8. Fast protein liquid chromatography (FPLC)

All FPLC equipment was purchased from Pharmacia. The AKTA FPLC working station

was controlled by UNICORN v. 4.0 software packet. Columns for anion exchange

chromatography (Q-Sepharose 26/10 or 16/10) and gel filtration (Superdex 75 130/10 and

HiPrep Desalt 26/10) were used.

8.5.1.9. Freeze-Drying

Lypholysation of protein was done in an Edwards freeze dryer Modulo (-40°C, 6 mbar).

8.5.1.10. Sodium dodecyl sulphate Polyacrylamide gel electrophoresis (SDS-PAGE)

The Acrylamide solutions were prepared according to Table 8.5. The separating gel was

pre-set between the plates for 30-45 min and then the stacking gel was pipetted in and

assembled within the 15 wells comb.

Ingredient 10 % Separating Gel (ml) 4 % Stacking Gel (ml) 40 % Acrylamide:Bis 37:1 2.5 1 Gel Buffer 3.33 2.5 50% glycerol in water 4.1 --- Distilled water --- 6.5 10 % AMPS 50 µl 50 µl TEMED 20 µl 10 µl TOTAL 10 ml 10 ml

Table 8.5 Stacking and Separating gels for SDS-PAGE.


147

Concentrated protein samples were directly added (2:1 v/v) to loading buffer. Diluted

samples (100 µl) were treated with trichloroacetic acid (10 µl, 72%, TCA) and incubated at

0 ˚C, 15 min. The resultant protein precipitate was pelleted by centrifugation (13,000 rpm,

5 min) and the supernatant discarded. The protein precipitate was resuspended in loading

buffer (5 µl) and TRIS base (2 M, 5 µl). The protein solution was then heated for 15 min at

100 ˚C in a Grant BT3 heat block to denature the protein and allow adhesion of SDS.

SDS-PAGE was run at constant current (80 mA). Buffers were prepared as follows:

Loading buffer: 9.456 g Trizma base, 1 ml mercaptoethanol, 1 g SDS, 10 ml glycerol,

0.01 g bromophenol blue, and made up to 100 ml with water, pH = 6.8.

Cathode buffer: 6.055 g Trizma base, 8.96 g Tricine, 0.5 g SDS made up to 500 ml with

distilled water, pH = 8.25.

Anode buffer: 12.11 g Trizma base, 1.5 g SDS to 500 ml with distilled water (pH 8.9).

Gel buffer: 181.7 g Trizma base and 1.5 g SDS to 500 ml with distilled water, pH = 8.4.

Staining solution: 2.5g/l Coomassie ble in 45% MeOH/45% glacial AcOH.

Destaining solution: 45% MeOH / 10% glacial AcOH in water.

8.5.2. Growth of E. coli, and expression and purification of ACP, ACPS and MCAT

E. coli BL21(DE3) competent cells were transformed with the appropriate plasmid for

expression of the corresponding ACP, ACPS or MCAT according to the supplier protocol

(Promega). They were then selected in carbenicillin-supplemented agar plates overnight. A

single colony was picked up using sterile toothpicks. This seed culture was amplified for 2-

4 h into at 37 ˚C, 220 rpm and used to inoculate the starter culture (LB medium, 200 ml).

The starter culture was grown until the optical density at 595 nm (A595) was 0.6, and then

centrifuged (10 min, 10,000 rpm).

Cells were re-suspended and then grown in 2 litres of LB medium (plus 100 µg/l

carbenicillin) at 37 ˚C, 250 rpm. IPTG (1 mM) was added when A595 was 0.6-0.8, and the

cultures incubated for further 4h at 32 ˚C, then harvested by centrifugation. Cells were

lysed by sonication and cell-free extracts subjected to the corresponding purification

protocol.


148

8.5.2.1. Purification of ACP

The purification of heterologous C17S and C17S/other mutant ACP has been carried out

following the procedure described by Crosby and Cox.52 Cell-free extracts were treated

with streptomycin sulphate (20 g/l) to precipitate DNA (9,000 rpm, 15 min). Ammonium

sulphate precipitation was then undertaken. First, 60% cut was performed and the pellet

(9,000 rpm, 15 min) discarded. The supernatant was subjected to a 100% cut and the pH

adjusted to 3.4. This solution was then stored at 4˚C for 48 hr to allow for complete

precipitation of ACP. Subsequent centrifugation (18,000 rpm, 20 min) caused a pellet

which was resuspended and dyalised (Spectra/Pore 3,000 Da cut molecular porous

membrane tubing, Spectrum Laboratories) against TRIS buffer (50 mM, pH = 8.0),

overnight.

This sample was then purified by anionic exchange fast protein liquid chromatography

(Q-Sepharose HiLoad 26/10 or 16/10). A NaCl gradient from 0 to 1 M was performed, the

apo-ACP peak observed at around 0.5 M salt, whereas holo-ACP (dimer) was eluted at

around 0.6 M NaCl. If the sample required further purification (as judged by SDS-PAGE),

it was injected into a Superdex 75 130/10 to separate proteins by size. The ACP-containing

fractions (SDS-PAGE) were desalted (HiPrep Desalt 26/10), flash-frozen in liquid nitrogen

and freeze-dried. The freeze-dried protein was weighed and dissolved in 100 mM phosphate

buffer, pH = 7.3. Yields of purification of holo-ACP were in the range 5-10 mg/l. Apo-ACP

was recovered in higher yields (30 mg/l).

8.5.2.2. Purification of S. coelicolor his6-ACPS and act his6-KR

Purification of S. coelicolor ACPS and act KR was carried out following the procedure of

Cox47 and Hadfield,32 respectively. Cell-free extracts were subjected to fractional

precipitation by streptomycin sulphate (20g/l) to precipitate DNA (9,000 rpm, 15 min). The

supernatant was then filtered (0.45 µm) and applied to a Nickel chromatography column

(NTA His-Bind resin -Novagen), which was run as described in Section 8.5.1.7. ACPS or

KR-containing fractions were immediately desalted by FPLC (HiPrep Desalt 26/60). The

desalted protein solution was flash-frozen in liquid nitrogen and stored at -80 ˚C.


149

8.5.2.3. Purification of S. coelicolor his6-MCAT

Purification of S. coelicolor MCAT was carried out following the procedure of

Szafranska.46 Cell-free extracts were subjected to fractional precipitation by streptomycin

sulphate (20g/l) to precipitate DNA (9,000 rpm, 15 min). The supernatant was then filtered

(0.45 µm) and applied to a Nickel chromatography column (NTA His-Bind resin -

Novagen), which was run as described in Section 8.5.1.7. MCAT-containing fractions were

subjected to further purification by anion exchange chromatography (Q-Sepharose HiLoad

26/10) and finally desalted by FPLC (HiPrep Desalt). The desalted protein solution was

flash-frozen in liquid nitrogen and stored at -20 ˚C.

8.5.3. Growth of S. coelicolor, expression and purification of his6-act-KSα /KSβ .

8.5.3.1. Growth of bacteria and expression of KSα/KSβ

The liquid medium for the growth of S. coelicolor A3(2) CH999 carrying the genes for

expression of wild type or mutant KSα/KSβ was Super-Yeme (SY) medium. 2 ml of frozen

mycelium in glycerol were amplified for 2-5 days at 30 ˚C, 220 rpm in SY medium

supplemented with kanamycin (100 µg/ml). This was used as starter culture for 2 litres of

SY medium (plus 100 µg/l kanamycin), which were subsequently grown at 30 ˚C, 220 rpm

for 2-3 days and then induced (thiostrepton, 10 µg/ml) and the culture incubated for a

further couple of days. Then the cultures were harvested (20x2 min, 9,000 rpm, diluted to

50% with water) and the cells lysed by sonication.

8.5.3.2. Purification of KSα/KSβ

Purification of KSα/KSβ was done essentially as described previously by the Bristol

group.8 Fractional precipitation by streptomycin sulphate was followed by

ultracentrifugation (43,000 rpm, 30 min). His6-(mutant)-KSα/KSβ was then purified by

nickel-affinity chromatography as described in 8.5.1.7. KSα/KSβ-containing fractions were


150

immediately desalted by FPLC (HiPrep Desalt). The desalted protein solution was flash-

frozen in liquid nitrogen and stored at -80 ˚C.

8.6. Protein characterization methods

8.6.1. Protein Quantification

8.6.1.1. Bradford Assay

The Bradford reagent was made up as follows: 10 mg Brilliant Blue G, 10 ml 85%

phosphoric acid, 5 ml 95% ethanol, up to 100 ml with distilled water, then filtered. A blank

sample was routinely tested (100 µl distilled water in 1 ml Bradford reagent), as well as

Bovine Serum Albumin (Sigma) standard samples for calibration (20 µl protein solution

and 80 µl distilled water in 1 ml Bradford reagent). Protein samples (KSα/KSβ and ACP)

were prepared in the same manner. All the samples were incubated for 5 min at room

temperature and then the absorbance at 595 nm was measured.

8.6.1.2. Bicinchonic Acid Assay (BCA)

The BCA Protein Assay Kit was purchased from Pierce. The BCA reagent was made up as

follows: 50 parts of reagent A (1%BCA-Na2, 2% Na2CO3, 0.16% Na2 tartrate, 0.4% NaOH

and 0.95 NaHCO3) were mixed with 1 part of reagent B (4% CuSO4.5 H2O). A blank

sample was routinely tested (20 µl distilled water in 1 ml BCA reagent), as well as Bovine

Serum Albumin (Sigma) standard samples for calibration. Protein samples (KSα/KSβ and

ACP) were prepared in the same manner. All the samples were incubated for 30 min at 60˚

C and the absorbance at 562 nm read.


151

8.6.1.3. Extinction coefficient of proteins.

The extinction coefficients at 280 nm for ACP (2400 M-1cm-1) and KSα/KSβ (84200 M-1

cm-1) have been previously determined within the group (Arthur, 2004). Protein

concentrations were calculated from Beer’s law:

A280 = ε c L

where A280 is the absorbance at a given wavelength, ε is the extinction coefficient, c

is the concentration of the protein and L is the pathlength of the UV beam through the

solution.

8.6.1.4. Electro-Spray Mass Spectrometry (ESMS)

In order to desalt and concentrate protein samples for analysis by ESMS, protein solutions

were prepared by hydrophobic interaction with a C4 resin as described by Winston and

Fitzgerald.185 C4 resin (3-5 mg, Jupiter 15u 300 Å, Phenomenex) was equilibrated in HPLC

grade methanol (100 µl) and the protein solution added. The sample was vortexed (5 min)

and centrifuged (13,000 rpm, 5 min). The supernatant was discarded, the pellet washed

twice with 0.1% aqueous trifluoroacetic acid (TFA, 1 ml), then vortexed and spun down

(13,000 rpm, 5 min). The final pellet was eluted in acetonitrile (25 µl).

ESMS analysis was performed on a QSTAR® XL system (Applied Biosystems, Foster

City, California) equipped with a chip based NanoESI source, Nanomate® 100 (Advion

biosciences, Ithaca NY) using Chipsoft software (version 5.1). The Nanomate® 100 source

was operated using a 400 nozzle chip, with a delivery pressure of 0.3 psi and spray voltage

of 1.40 kV. The QSTAR® XL was controlled using AnalystTM QS software (version 1.1) in

positive TOF MS mode.

8.7. Protein assays.

All assays were performed in phosphate buffer (pH = 7.3) at 30 ˚C, unless otherwise

described.


152

8.7.1. Phosphopantetheninylation of E47V apo-ACP to produce E47V holo-ACP.

ACPS was incubated with E47V apo-ACP and CoA in a 1:10:100 ratio in TRIS buffer, pH

= 8.8 in the presence of MgCl2 (10 mM). The reaction was monitored by ESMS and

allowed for 12 h. Purification of holo-ACP from ACPS and unreacted apo-ACP was

achieved by anion-exchange chromatography using a gradient of NaCl 0-1 M as described

in Section 8.5.2.1.

8.7.2. Reduction of ACP dimers.

Holo-ACP monomer was produced after reduction of the disulfide bonds between PP arms

of two ACP by a sulphydryl reducing agent. ACP (50-500 µM) was treated for 1 h, 30 ˚C

with either tris(2-carboxyethyl)phosphine (TCEP) or dithiothreitol (DTT), at a final

concentration of 1-2 mM. ESMS of ACP species was then performed as described in

Section 8.6.1.4.

8.7.3. Acylation of holo-ACP

Following the procedure of Hitchmann, acetyl and malonyl-ACP were produced by

incubation of ACP (500 µM) with either 1-acetylimidazole (5 mM) or malonyl CoA (2 mM)

for 2-4 h, and the reactions monitored by ESMS.

8.7.4. Study of the self-malonylation of ACP using α-ketoglutarate dehydrogenase

(KGDH)

The kinetic study of self-malonylation of ACP by the α-ketoglutarate dehydrogenase

(KGDH) coupling system was carried out by measuring the change in A340 due to the

absorbance of NADH. All enzymatic reactions were in a final volume of 200 µl and

performed in 96-well microplates in the plate reader. Assays were performed essentially as

described in the literature. The final concentrations of each ingredient in the assay were as


153

follows: 100 mM phosphate buffer pH = 7.3, 1 mM EDTA, 1 mM TCEP, 2 mM α-

ketoglutaric acid (KGA), 0.5 mM NAD+, 0.4 mM thiamine pyrophosphate (TPP), 1 mM

MgCl2 and 80 mU/ 100 µl KGDH. A working stock of 10 times their final concentrations

of EDTA, TCEP, KGA, TPP and NAD+ was made up. Assays were started by addition of

monomer holo-ACP and initial rates approximated from the linear regressions of the

absorbance traces during the first 0.5 to 2 min (6 to 24 time points).

8.7.5. Minimal actinorhodin polyketide synthase assays and HPLC analysis of SEK4

and SEK4b

All minimal PKS assays were performed in 100 mM phosphate buffer, pH = 7.3 in the

presence of 2 mM EDTA and 1 mM TCEP at 30 ˚C. The protocol for production of

polyketides by the act minimal PKS was adapted from Matharu.8 In general, malonyl CoA

was added to the reaction buffer prior to addition of KSα/KSβ. The enzyme was

equilibrated for 1 min under reaction conditions and assays were then started by addition of

monomer holo-ACP.

At designated time points, aliquots (50-100 µl) were taken from the general reaction

volume (up to 1 ml), acidified (NaH2PO4, 500 mg/ml) and extracted with 2 x 500 µl ethyl

acetate, as described by Matharu.8 A gentle stream of nitrogen helped evaporating the

organic solvent. The concentrated products were re-suspended in methanol (50 µl) and

injected into the HPLC system.

Analysis of polyketides was performed in a Dionex HPLC equipped with a pump

system P680, a Thermosthated Column Compartment TCC-100 and a PDA-100 Photodiode

Array Detector. The column used for detection and quantitative analysis of polyketides was

a Luna 5u C18 250 x 4.60 mm (Phenomenex), thermosthated at 25 ˚C. The mobile phase

for RP-HPLC was water/acetonitrile. A gradient from 5 to 75% acetonitrile was carried out

over 35 min at 1 ml/min, followed by washing (95% acetonitrile) and re-equilibration (95%

H2O) steps. SEK4 was detected after 19.6 min and SEK4b after 20.3 min. A slight

difference in retention times was observed when a Luna 5u C18(2) by Phenomenex was

used. The elution volume of SEK4 was 19.1 min; SEK4b was detected after 19.8 min.


154

8.7.6. Kinetic studies on the actinorhodin minimal polyketide synthase

Kinetics of the actinorhodin minimal polyketide synthase were studied in a Spectramax 190

plate reader (Molecular devices), by monitoring the absorbance of the polyketides

produced.

8.7.6.1. Calibration of the method

Solutions of purified SEK4 and SEK4b were used as standards for calibration. Working

solutions of 0.1 mg/ml (314 µM) SEK4 and 1 mg/ml (3.14 mM) SEK4b in 1:1 water:

acetonitrile were made up, and diluted in 100 mM phosphate buffer, pH = 7.3 to the desired

concentrations in a total volume of 200 µl. The absorbance at 293 nm was read and the

calibration curve (1-100 µM) constructed. The experimental error was estimated as less

than 7% by repeating the calibration procedure with three independent series.

8.7.6.2. Minimal polyketide synthase assays to measure self-malonylation of holo-

ACP

All enzymatic reactions were in a final volume of 200 µl and performed in an absorbance

plate reader. The final concentrations of all components were 100 mM phosphate buffer, 2

mM EDTA, 1 mM TCEP, 5 to 1000 µM malonyl CoA, 3 µM KSα/KSβ and 0.2 to 160 µM

holo-ACP. KSα/KSβ was incubated for 2 minutes in the reaction mixture containing

malonyl CoA, EDTA and TCEP, which was pre-equilibrated at 30 ˚C in the plate reader.

Assays were started by addition of monomer holo-ACP. The absorbance of the samples

was immediately measured for 3-20 minutes, every 4 seconds. Initial reaction rates were

calculated from the linear regressions of the absorbance traces during the linear period,

usually 1 to 5 minutes (15 to 60 time points).


155

8.7.6.3. Minimal polyketide synthase assays to measure inhibition of self-

malonylation of holo-ACP by CoA-SH

CoA-SH was purchased from Sigma, aliquoted in 100 mM fractions and kept at -20 ˚C. The

concentration of CoA-SH in inhibition assays ranged from 500 to 2000 µM. Concentrations

of holo-ACP and KSα/KSβ were 50 µM and 3 µM respectively.

8.7.6.4. Minimal polyketide synthase assays to measure the kinetics of KSβ

To study the kinetics of KSβ, S. coelicolor MCAT (0.5-2.5 µM) was added to the reaction

mixture to accelerate the malonylation of ACP. The concentration of KSα/KSβ in these

assays was 0.2-0.5 µM. ACP concentrations were varied as needed for the determination of

kinetic parameters. MCAT-supplemented act minimal assays were then carried out as

described in Section 8.7.6.2.

8.7.6.5. Minimal polyketide synthase assays to measure the kinetics of KSα

In order to study the kinetics of KSα, acetyl-ACP was added to a concentration of 30 µM,

and assays then started by addition of malonyl CoA (1 mM). MCAT was added in

concentrations of 0.5 µM. Holo-ACP concentration in the assays was about 5 to 10-fold the

KM value for the corresponding ACP species.For the rest, assays were executed as

described in Section 8.7.6.2.

8.7.6.6. Minimal polyketide synthase assays to measure inhibition of KSα/KSβ by

apo-ACP

Assays were carried out as described in Section 8.7.6.2. MCAT concentration was 0.5 µM.

KSα/KSβ concentration was 0.25 µM. Holo-ACP concentration was 20 µM. Apo-ACP was

added in concentrations of 25-200 µM.


156

8.7.6.7. Minimal polyketide synthase assays to measure the kinetics of CA KSα/KSβ

To study the kinetics of CA KSα/KSβ, S. coelicolor MCAT (0.5-2.5 µM) was added to

assays in which the concentration of mutant KSα/KSβ was 1-2 µM. and assays were then

carried out as described in Section 8.7.6.2.

8.7.6.8. Data analysis

Initial reaction rates were deduced from absorbance data by fitting to a straight line using

the software provided by the manufacturer of the plate reader (Molecular Devices). In

general, assays were done at least in triplicate, and the average value is shown throughout

this thesis as a mean plus or minus an error.

For the determination of Michaelis-Menten parameters, these data were used to produce

plots showing the hyperbolic dependence of reaction rates on substrate concentration.

Kinetic parameters were then calculated by fitting this plots to a hyperbolic function using

Sigmaplot 8TM.

Hanes linear plots were also produced to assess the quality of the kinetic data. Hanes

plots result from Eq. 4 (see Section 1.4.1), with some re-arrangement:

!

[ S ] / rate =KM

kcat [ Eo]+

1

kcat [ Eo][ S ] (Eq. 8)

This means that a plot of [S] / rate vs. [S] should be a straight line. This plot was used as it

is recommended by modern enzyme kinetics textbooks as the less biased linear treatment of

kinetic data.94


157

1 Mann, J. (1987) Secondary Metabolism, 2nd edition, Clarendon Press, Oxford.

2 Luckner, M. (1990) Secondary metabolism in microorganism, plants and animals,

3rd edition, Springer-Verlag, Berlin.

3 Staunton, J., and Weissman, K. J. (2001) Polyketide Biosynthesis: a millennium

review, Nat Prod Rep. 18, 380-416.

4 Cox, R. J. (2002) Biosynthesis, Annu Rep Prog Chem, Sect. B, 98, 223-251.

5 Cox, R. J. (2007) Polyketides, proteins and genes in fungi: programmed nano-

machines begin to reveal their secrets, Org Biomol Chem. 5, 2010-2026.

6 Kieser, T., Bibb, M.J., Buttner, M.J., Chater, K.F. and Hopwood, D.A. (2000)

Practical Streptomyces genetics, The John Innes Foundation, Chichester.

7 Richardson, M. and Khosla, C. (1999) Comprehensible Natural Products Chemistry

v.1. Elsevier.

8 Matharu, A.L., Cox, R.J., Crosby, J., Byrom, K.J. and Simpson, T.J. (1998) MCAT

is not required for in vitro polyketide synthesis in a minimal actinorhodin

polyketide synthase from Streptomyces coelicolor, Chem Biol 5, 699-711.

9 Wohlert, S., Wendt-Pienkowski, E., Bao, W. and Hutchinson, C.R. (2001)

Production of Aromatic Minimal Polyketides by the Daunorubicin Polyketide

Synthase Genes Reveals the Incompatibility of the Heterologous DpsY and JadI

Cyclases, J Nat Prod. 64, 1077-1080.

10 Taguchi, T., Ebizuka, Y., Hopwood, D.A. and Ichinose, K. (2001) A new mode of

stereochemical control revealed by analysis of the biosynthesis of dihydrogranaticin

in Streptomyces violaceoruber Tu22, J Am Chem Soc. 123, 11376-11380.

11 Yu, T-W., Bibb, M. J., Revill, W. P. and Hopwood, D. A. (1994) Cloning,

Sequencing, and Analysis of the Griseusin Polyketide Synthase Gene Cluster from

Streptomyces griseus, J Bacteriol 176, 2627-2634.

12 Räty, K., Kantola, J., Hautala J., Hakala, J., Ylihonko, K. and Mäntsälä, P. (2002)

Cloning and characterization of Streptomyces galilaeus aclacinomycins polyketide

synthase (PKS) cluster, Gene 293, 115-122.

13 Meadows E.S. and Khosla,C. (2001) In Vitro Reconstitution and Analysis of the

Chain Initiating Enzymes of the R1128 Polyketide Synthase, Biochemistry 40,

14855-14861.

14 Petkovi, H., Thamchaipenet, A., Zhou, L., Hranueli� , D., Raspor, P., Waterman,

P.G., and Hunter, I.S. (1999) Disruption of an aromatase/cyclase from the


158

oxytetracycline gene cluster of Streptomyces rimosus results in production of novel

polyketides with shorter chain lengths, J Biol Chem. 274, 32829-32834.

15 Piel, J., Hertweck, C., Shipley, P. R., Hunt, D. M., Newman, M. S., and Moore, B.

S. (2000) Cloning, sequencing and analysis of the enterocin biosynthesis gene

cluster from the marine isolate Streptomyces maritimus: evidence for the derailment

of an aromatic polyketide synthase, Chem Biol. 7, 943–955.

16 Bentley, S.D., Chater, K.F., Cerdeno-Tarraga, A.M., Challis, G.L., Thomson, N.R.,

James, K.D., Harris, D.E., Quail, M.A., Kieser, H., Harper, D., Bateman, A.,

Brown, S., Chandra, G., Chen, C.W., Collins, M., Cronin, A., Fraser, A., Goble, A.,

Hidalgo, J., Hornsby, T., Howarth, S., Huang, C.H., Kieser, T., Larke, L., Murphy,

L., Oliver, K., O'Neil, S., Rabbinowitsch, E., Rajandream, M.A., Rutherford, K.,

Rutter, S., Seeger, K., Saunders, D., Sharp, S., Squares, R., Squares, S., Taylor, K.,

Warren, T., Wietzorrek, A., Woodward, J., Barrell, B.G., Parkhill, J. and Hopwood

D.A. (2002) Complete genome sequence of the model actinomycete Streptomyces

coelicolor A3(2), Nature 417, 141-147.

17 Rudd, B.A and Hopwood, D. A. (1979) Genetics of actinorhodin biosynthesis by

Streptomyces coelicolor A3(2), J Gen Microbiol. 114, 35-43.

18 Malpartida, F. and Hopwood, D. A. (1984) Molecular-Cloning of the whole

Biosynthetic-Pathway of a Streptomyces Antibiotic and its Expression in a

Heterologous Host, Nature 309, 462-464

19 Hallam, S. E., Malpartida, F. and Hopwood, D. A. (1988) Nucleotide sequence,

transcription and deduced function of a gene involved in polyketide antibiotic

synthesis in Streptomyces coelicolor, Gene 74, 305-320.

20 Fernandez-Moreno, M. A., Martinez, E., Boto, L., Hopwood, D. A. and Malpartida,

F. (1992) Nucleotide-Sequence and Deduced Functions of a Set of Co-transcribed

Genes of Streptomyces coelicolor A3(2), including the Polyketide Synthase for the

Antibiotic Actinorhodin, J Biol Chem. 267, 19278-19290.

21 Hopwood, D.A. (1997) Genetic contributions to understanding polyketide

synthases, Chem Rev. 97, 2465-2497.

22 McDaniel, R., Ebert-Khosla, S., Hopwood, D.A. and Khosla, C. (1993) Engineered

biosynthesis of novel polyketides, Science 262, 1546-1550.

23 McDaniel, R., Ebert-Khosla, S., Hopwood, D. A. and Khosla, C. (1994) Engineered

biosynthesis of novel polyketides: influence of a downstream enzyme on the


159

catalytic specificity of a minimal aromatic polyketide synthase, P Natl Acad Sci

U.S.A. 91, 11542-11546.

24 Fu, H., Ebert-Khosla, S., Hopwood, D.A. and Khosla, C. (1994) Engineered

Biosynthesis of Novel Polyketides: Dissection of the Catalytic Specificity of the act

Ketoreductase, J Am Chem Soc. 116, 4166-4170.

25 McDaniel, R., Evert-Khosla, S., Hopwood, D. A. and Khosla, C. (1994) Engineered

Biosynthesis of Novel Polyketides: actVII and actIV Genes Encode Aromatase and

Cyclase Enzymes, Respectively, J Am Chem Soc. 116, 10855-10859.

26 Valton, J., Fontecave, M., Douki, T., Kendrew, S.G and Niviere, V. (2006) An

aromatic hydroxylation reaction catalyzed by a two-component FMN-dependent

monooxygenase, J Biol Chem. 281, 27-35.

27 Taguchi, T., Kunieda, K., Takeda-Shitaka, M., Takaya, D., Kawano, N., Kimberley,

M.R., Booker-Milburn, K.I., Stephenson, G.R., Umeyama, H., Ebizuka, Y. and

Ichinose, K. (2004) Remarkably different structures and reaction mechanisms of

ketoreductases for the opposite stereochemical control in the biosynthesis of BIQ

antibiotics, Bioorg Med Chem. 12, 5917-5927.

28 Taguchi, T., Okamoto, S., Lezhava, A., Li, A., Ochi, Y. Ebizuka, Y. and Ichinose,

K. (2007) Possible involvement of ActVI-ORFA in transcriptional regulation of

actVI tailoring-step genes for actinorhodin biosynthesis, FEMS Microbio Lett. 269,

234-239.

29 Booker-Milburn, K.I., Gillan, R., Kimberley, M., Taguchi, T., Ichinose, K.,

Stephenson, G.R., Ebizuka, Y. and Hopwood, D.A. (2005) Enantioselective

reduction of β-keto acids with engineered Streptomyces coelicolor, Angew Chem Int

Edit. 44, 1121-1125.

30 Crump, M.P., Dempsey, C.E., Parkinson, J.A., Murray, M., Hopwood, D.A. and

Simpson, T.J. (1997) Solution structure of the actinorhodin polyketide synthase acyl

carrier protein from Streptomyces coelicolor A3(2), Biochemistry 36, 6000-60088.

31 Keating-Clay, A.T., Maltby, D.A., Medzihradszky K.F., Khosla, C. and Stroud,

R.M. (2004) An antibiotic factory caught in action, Nat Struc Mol Biol. 11, 888-

893.

32 Hadfield, A.T., Limpkin, C., Teartasin, W., Simpson, T.J., Crosby, J. and Crump,

M. (2004) The crystal structure of the actIII actinorhodin polyketide reductase:

proposed mechanism for ACP and polyketide binding, Structure 12, 1865-1875.


160

33 Sciara, G., Kendrew, S.G., Miele, A.E., Marsh, N.G., Federici, L., Malatesta, F.,

Schimperna, G., Savino, C. and Vallone, B. (2003) The structure of ActVA-Orf6, a

novel type of monooxygenase involved in actinorhodin biosynthesis, EMBO J. 22,

205.

34 Fu, H., Hopwood, D.A. and Khosla, C. (1994) Engineered biosynthesis of novel

polyketides: evidence for temporal, but not regiospecific, control of cyclization of

an aromatic polyketide precursor, Chem Biol. 1, 205-210.

35 McDaniel, R., Ebert-Khosla, S., Hopwood, D.A. and Khosla, C. (1993) Engineered

biosynthesis of novel polyketides, Science 262, 1546-1550.

36 Holak, T.A., Kearsley, S.K., Kim, Y. and Prestegard, J.H (1988) Three-dimensional

structure of acyl carrier protein determined by NMR pseudoenergy and distance

geometry calculations, Biochemistry 27, 6135-6142.

37 Xu, G-Y., Tam, A., Lin, L., Hixon, J., Fritz, C.C. and Powers, R (2001) Solution

structure of B. subtilis acyl carrier protein, Structure 9, 277-287.

38 Wong, H.C., Liu, G., Zhang, Y., Rock, C.O. and Zheng, J. (2002) The solution

structure of acyl carrier protein from Mycobacterium tuberculosis, J Biol Chem.

277, 15874-15880.

39 Li, Q., Khosla, C., Puglisi, J.D and Liu, C.W. (2003) Solution structure and

backbone dynamics of the holo form of the frenolicin acyl carrier protein,

Biochemistry 42, 4648-4657.

40 Kim, Y. and Prestegard, J.H. (1990) Demostration of a conformational equilibrium

in acyl carrier protein from spinach using rotating frame nuclear magnetic

resonance spectrometry, J Am Chem Soc. 112, 3707-3709.

41 Sharma, A.K., Sharma, S.K., Surolia, A., Surolia, N. and Sarma, S.P. (2006)

Solution structures of conformationally equilibrium forms of holo-acyl carrier

protein (PfACP) from Plasmodium falciparum provides insight into the mechanism

of activation of ACPs, Biochemistry 45, 6904-6916.

42 Cox, R. J., Crosby, J., Daltrop, O., Glod, F., Jarzabek, M., Nicholson, T. P,

Simpson, T. J., Soulas, F. and Westcott, J. (2002) Cloning, expression and substrate

activity of Streptomyces coelicolor holo-Acyl Carrier Protein synthase, J. Chem.

Soc., Perkin Trans 1, 2002.


161

43 Revill, W. P., Bibb, M. J. and Hopwood, D. A. (1995) Purification of a

malonyltransferase from Streptomyces coelicolor A3(2) and analysis of its genetic

determinant, J Bacteriol. 177, 3946-3952.

44 Summers, R. G., Ali, A., Shen, B., Wessel, W. A. and Hutchinson, C. R. (1995)

Malonyl-coenzyme A:acyl carrier protein acyltransferase of Streptomyces

glaucescens: a possible link between fatty acid and polyketide biosynthesis,


45 Keatinge-Clay, A. T., Shelat, A. A., Savage, D. F., Tsai, S. C., Miercke, L. J.,

O'Connell, J. D. 3rd, Khosla, C. and Stroud, R. M. (2003) Catalysis, specificity, and

ACP docking site of Streptomyces coelicolor malonyl-CoA:ACP transacylase,

Structure 11, 147-154.

46 Szafranska, A.E., Hitchman T.S., Cox, R.J., Crosby, J. and Simpson, T.J. (2002)

Kinetic and mechanistic analysis of the malonyl CoA: ACP transacylase from

Streptomyces coelicolor indicates a single catalytically competent serine

nucleophile at the active site, Biochemistry 41, 1421-1427.

47 Cox, R.J., Hitchman, T.S., Byrom, K.J., Findlow, S.C., Tanner, J.A., Crosby, J. and

Simpson, T.J. (1997) Post-translational modification of heterologously expressed

Streptomyces Type II polyketide synthase acyl carrier proteins, FEBS Lett. 405,

267-272.

48 Haapalainen, A.M., Merilainen, G. and Wierenga, R.K. (2006) The thiolase

superfamily: condensing enzymes with diverse reaction specificities, Trends

Biochem Sci. 31, 64-71.

49 Zhang, Y-M., Hurlbert, J., White, S. W., and Rock, C. O. (2006) Roles of the

Active Site Water, Histidine 303, and Phenylalanine 396 in the Catalytic

Mechanism of the Elongation Condensing Enzyme of Streptococcus pneumoniae, J

Biol Chem. 281, 17390-17399.

50 Price, A.C., Choi, K-H., Heath, R.J., Li, Z., White, S.W. and Rock, C.O. (2001)

Inhibition of β-ketoacyl-acyl carrier protein synthases by thiolactomycin and

cerulenin. Structure and mechanism, J Biol Chem. 276, 6551-6559.

51 Olsen, J.G., Kadziola, A., von Wettstein-Knowles, P., Siggaard-Andersen, M. and

Larsen, S. (2001) Structures of β-ketoacyl-acyl carrier protein synthase I complexed

with fatty acids elucidate its catalytic machinery, Structure 9, 233-243.


162

52 Crosby, J., Byrom, K., Hitchman, T.S., Cox, R.J., Crump, M.P., Findlow, S.C.,

Bibb, M.J. and Simpson, T.S. (1998) Acylation of Streptomyces Type II polyketide

synthase acyl carrier proteins, FEBS Lett. 433, 132-138.

53 Carreras, C.W. and Khosla, C. (1998) Purification and in Vitro Reconstitution of the

Essential Protein Components of an Aromatic Polyketide Synthase, Biochemistry

37, 2084.

54 Tang, Y., Tsai, S.C. and Khosla, C. (2003) Polyketide chain length control by chain

length factor, J Am Chem Soc. 125, 12708-12709.

55 Hitchman, T.S., Crosby, J., Byrom, K.J., Cox, R.J. and Simpson, T.J. (1998)

Catalytic self-acylation of type II polyketide synthase acyl carrier proteins, Chem

Biol. 5, 35-47.

56 Dreier, J., Shah, A.N. and Khosla, C. (1999) Kinetic analysis of the actinorhodin

aromatic polyketide synthase, J Biol Chem. 274, 25108-25112.

57 Zhou, P., Florova, G., and Reynolds, K.A. (1999) Polyketide synthase acyl carrier

protein (ACP) as a substrate and a catalyst for malonyl ACP biosynthesis, Chem

Biol. 6, 577-584.

58 Florova, G., Kazanina, G. and Reynolds, K.A. (2002) Enzymes involved in fatty

acid and polyketide biosynthesis in Streptomyces glaucescens: role of FabH and

FabD and their acyl carrier protein specificity Biochemistry 41, 10462-10471.

59 Arthur, C.J., Szafranska, A., Evans, S.E., Findlow, S.C., Burston, S.G., Owen, P.,

Clark-Lewis, I., Simpson, J., Crosby, J. and Crump, M.P. (2005) Self-malonylation

is an intrinsic property of a chemically synthesized type II polyketide synthase acyl

carrier protein, Biochemistry 44, 15414-15421.

60 Arthur, C.J., Szafranska, A., Long, J., Mills, J., Cox, R.J., Findlow, S.C., Simpson,

T.J., Crump, M.P. and Crosby, J. (2006) The malonyl transfer activity of Type II

PKS acyl carrier proteins, Chem Biol. 13, 587-596.

61 Misra, A., Sharma, S. K., Surolia, N. and Surolia, A. (2007) Self-Acylation

Properties of Type II Fatty Acid Biosynthesis Acyl Carrier Protein, Chem Biol. 14,

775-783.

62 Bisang, C., Long, P.F., Cortes, J., Westcott, J., Crosby, J., Matharu, A.L., Cox, R.J.,

Simpson, T.J., Staunton, J. and Leadlay, P.F. (1999) A chain initiation factor

common to both modular and aromatic polyketide synthases, Nature 401, 502-505.


163

63 Tsai, S-C. (2004) A Fine Balancing Act of Type III Polyketide Synthases, Chem

Biol. 11, 1174-1175.

64 Gryschow, S., Buchholz, T. J., Seufert, W., Dordick, J. S. and Sherman, D. H.

(2007) Substrate Profile Analysis and ACP-Mediated Acyl Transfer in Streptomyces

coelicolor Type III Polyketide Synthases, ChemBioChem 8, 863-868.

65 Austin, M. B. and Noel, J.P. (2003) The chalcone synthase superfamily of type III

polyketide synthases, Nat Prod Rep. 20, 79–110.

66 Katsuyama, Y. Matsuzawa, M. Funa, N. and Horinouchi, S. (2007) In Vitro

Synthesis Of Curcuminoids By Type III Polyketide Synthase From Oryza Sativa, J

Biol Chem. 282, 37702-37709.

67 Jez, J. M., Ferrer, J. L., Bowman, M. E., Dixon, R.A and Noel, J.P. (2000) Structure

of chalcone synthase and the molecular basis of plant polyketide biosynthesis, Nat

Struc Biol. 6, 775-785.

68 Austin, M. B., Bowman, M.E., Ferrer, J. L., Schröder, J. and Noel, J.P. (2004) An

aldol switch discovered in stilbene synthases mediates cyclization specificity of type

III polyketide synthases, Chem Biol. 11, 1179-1194. 69 Shomura, Y., Torayama, I., Suh, D. Y., Xiang, T., Kita, A., Sankawa, U., Miki, K.

(2005) Crystal structure of stilbene synthase from Arachis hypogaea, Proteins 60,

803-806.

70 Hopwood, D.A. (1997) Genetic contributions to understanding polyketides

synthases, Chem Rev. 97, 2465-2497.

71 Hill, A.M. (2006) The biosynthesis, molecular genetics and enzymology of the

polyketide-derived metabolites, Nat Prod Rep. 23, 256-320.

72 McDaniel, R., Welch, M. and Hutchinson, C.R. (2005) Genetic approaches to

polyketide antibiotics 1, Chem Rev. 105, 543-558.

73 Khosla, C., Tang, Y., Chen, A. Y., Schnarr, N. and Cane, D. (2007) Structure and

mechanism of the 6-Deoxyerythronolide B Synthase, Annu Rev Biochem 76, 11.1-

11.27

74 Townsend, C. A., Crawford, J. M and Bililign, T. (2006) New images evoke

FAScinating questions, Chem Biol. 13, 349-351.

75 Wakil, S. J., Pugh, E. L., and Sauer, F. (1964) The Mechanism of Fatty Acid

Synthesis, P Natl Acad Sci U. S. A. 52, 106-114.


164

76 Stoops, J.K., Arslanian, A.W., Oh, Y.H., Aune, K.C., Vanaman, T.C., and Wakil,

S.J. (1975) Presence of Two Polypeptide Chains comprising Fatty Acid Synthetase,

P Natl Acad Sci U. S. A. 75, 1940-1944.

77 Stoops, J. K and Wakil, S. J. (1981) Animal Fatty Acid Synthetase: A Novel

Arrangement of the β-Ketoacyl Synthetase Sites Comprising Domains of the Two

Subunits, J Biol Chem. 256, 5128-5133.

78 Stoops, J. K., Wakil, S. J., Uberbacherl, E. C., and Bunick, G. J. (1987) Small-angle

Neutron-scattering and Electron Microscope Studies of the Chicken Liver Fatty

Acid Synthase, J Biol Chem. 262, 10246-10251.

79 Joshi, A. K., Witkowsky, A. and Smith, S. (1997) Mapping of functional

interactions between domains of the animal fatty acid synthase by mutant

complementation in vitro, Biochemistry 36, 2316-2322.

80 Joshi, A. K., Witkowsky, A. and Smith, S. (1998) The Malonyl/Acetyltransferase

and β-Ketoacyl Synthase Domains of the Animal Fatty Acid Synthase Can

Cooperate with the Acyl Carrier Protein Domain of Either Subunit, Biochemistry

37, 2515-2523.

81 Smith, S. and Tsai, S-C (2007) The type I fatty acid and polyketide synthases: a tale

of two megasynthases, Nat Prod Rep. 24, 1041-1072.

82 Witkowski, A., Joshi, A. K., Rangan, V. S., Falick, A. M., Witkowska, H. E. and

Smith, S. (1998) Dibromopropanone Cross-linking of the Phosphopantetheine and

Active-site Cysteine Thiols of the Animal Fatty Acid Synthase Can Occur Both

Inter- and Intrasubunit, J Biol Chem 274, 11557-11563.

83 Chirala S. S. and Wakil S. J. (2004) Structure and Function of Animal Fatty Acid

Synthase, Lipids 39, 1045-1053.

84 Smith, S., Witkowski, A. and Joshi, A. K. (2003) Structural and functional

organization of the animal fatty acid synthase, Prog Lipid Res. 42, 289-317.

85 Maier, T., Jenni, S and Ban, N. (2006) Architecture of the mammalian fatty acid

synthase at 4.5 Å resolution, Science 311, 1258-1262.

86 Asturias, F. J., Chadick, J. Z., Cheung, I. K., Stark, H., Witkowski, A., Joshi, A. K.

and Smith, S. (2005) Structural and functional organization of the animal fatty acid

synthase, Nat Struc Mol Biol. 12, 225–232.


165

87 Tang, Y., Kim, C.Y., Mathews, I.I., Cane, D.E. and Khosla, C. (2006) The 2.7-Å

crystal structure of a 194-kDa homodimeric fragment of the 6-deoxyerythronolide B

synthase. P Natl Acad Sci U. S. A. 103, 11124-11129.

88 Keatinge-Clay, A.T. and Stroud R.M. (2006) The Structure of a Ketoreductase

Determines the Organization of the β-Carbon Processing Enzymes of Modular

Polyketide Synthases, Structure 14, 737-748.

89 Tsai, S.-C., Lu, H., Cane, D.E., Khosla, C. and Stroud, R.M. (2003) Insights into

channel architecture and substrate specificity from crystal structures of two

macrocycle-forming thioesterases of modular polyketide synthases, Biochemistry

41,12598-12606.

90 Tsai, S.C., Miercke, L.J., Krucinski, J., Gokhale, R., Chen, J.C., Foster, P.G., Cane,

D.E., Khosla, C. and Stroud, R.M. (2001) Crystal structure of the macrocycle-

forming thioesterase domain of the erythromycin polyketide synthase: versatility

from a unique substrate channel, P Natl Acad Sci U. S. A. 98,14808-14813.

91 Broadhurst, R.W., Nietlispach, D., Wheatcroft, M.P., Leadlay, P.F. and Weissman,

K.J. (2003) The structure of docking domains in modular polyketide synthases,

Chem Biol. 10, 723-731.

92 Alekseyev, V.V., Liu, C. W., Cane, D. E., Puglisi, J. D. and Khosla, C. (2007)

Solution structure and proposed domain–domain recognition interface of an acyl

carrier protein domain from a modular polyketide synthase, Protein Sci. 16, 2093-

2107.

93 Buc, J. and Giordani, R. (1998) A spectrophotometric method for kinetic studies

with quinone-dependent oxidoreductases. Application to detection in membranes of

nitrate reductase activity with menadione and duroquinone as electron donors

Enzyme Microb Tech. 22, 165-169.

94 Cornish-Bowden, A. (2004) Fundamentals of Enzyme Kinetics, 3rd ed. Portland

Press, London.

95 Branden, C. and Tooze, J. (1999) Introduction to Protein Structure, 2nd ed, Garland

Publishing Inc., New York.

96 Nicholson, T.P., Winfield, C., Westcott, J., Crosby, J., Simpson, T.J. and Cox, R.J.

(2004) First in vitro directed biosynthesis of new compounds by a minimal Type II

polyketide synthase: evidence for the mechanism of chain determination, Chem.

Comm. 6, 686-687.


166

97 Beltran-Alvarez, P., Cox, R. J., Crosby, J. and Simpson, T. J. (2007) Dissecting the

component reactions catalyzed by the actinorhodin minimal polyketide synthase,


98 Tang, Y., Lee, T-S., Kobayashi, S. and Khosla, C. (2003) Ketosynthases in the

initiation and elongation modules of aromatic polyketide synthases have orthogonal

acyl carrier protein specificity, Biochemistry 42, 6588-6595.

99 Lee, T-S., Khosla, C. and Tang, Y. (2005) Orthogonal protein interactions in spore

pigment producing and antibiotic producing polyketide synthases, J Antibiot. 58,

663-666.

100 Wu, N., Cane, D. E. and Khosla, C. (2002) Quantitative analysis of the relative

contributions of donor acyl carrier proteins, acceptor ketosynthases, and linker

regions to intermodular transfer of intermediates in hybrid polyketide synthases,


101 Mathews, D.K. and van Holde, K.E. (2000) Biochemistry, 2nd ed. McGraw-Hill,

Boston.

102 Holak, T. A., Kearsley, S.K., Kim, Y. and Prestegard, J. H. (1998) Three-

dimensional structure of acyl carrier protein determined by NMR pseudoenergy and

distance geometry calculations, Biochemistry 27, 6135-6142.

103 Olsen, J. G., Kadziola, A., Wettstein-Knowles, P., Siggaard-Andersen, M.,

Lindquist, Y., and Larsen, S. (1999) The X-ray crystal structure of β-ketoacyl [acyl

carrier protein] synthase I, FEBS Lett. 460, 46–52

104 Serre, L., Swenson, L., Green, R., Wei, Y., Verwoert, I.I.G.S., Verbree, E.C.,

Stuitje, A.R. and Derewenda, Z.S. (1994) Crystallization of the malonyl coenzyme

A-acyl carrier protein transacylase from Escherichia coli. J Mol Biol. 242, 99-102.

105 Serre, L., Verbree, E.C., Dauter, Z., Stuitje, A.R. and Derewenda, Z.S. (1995) The

Escherichia coli malonyl-CoA:acyl carrier protein transacylase at 1.5-Å resolution.

Crystal structure of a fatty acid synthase component, J Biol Chem. 270, 12961-

12964.

106 Moche, M., Schneider, G., Edwards, P., Dehesh, K. and Lindqvist, Y. (1999)

Structure of the complex between the antibiotic cerulenin and its target, β-ketoacyl-

acyl carrier protein synthase, J Biol Chem. 274, 6031-6034.


167

107 Huang W., Jia J., Edwards P., Dehesh K., Schneider G. and Lindqvist Y. (1998)

Crystal structure of β-ketoacyl-acyl carrier protein synthase II from E.coli reveals

the molecular architecture of condensing enzymes, EMBO J. 17, 1183-1191.

108 Davies, C., Health, R.J., White, S.W. and Rock, C.O (2000) The 1.8 Å crystal

structure and active-site architecture of β-ketoacyl-acyl carrier protein synthase III

(FabH) from Escherichia coli, Structure 8, 185-195.

109 Price, A. C., Zhang, Y. M., Rock, C. O. and White, S. W. (2001) Structure of β-

ketoacyl-acyl carrier protein reductase from Escherichia coli: negative cooperativity

and its structural basis, Biochemistry 40, 417-428.

110 Baldock C., Rafferty J. B., Stuitje A. R., Slabas A. R. and Rice D. W. (1998) The

X-ray structure of Escherichia coli enoyl reductase with bound NAD+ at 2.1 Å

resolution, J Mol Biol. 284, 1529-46.

111 Leesong, M., Henderson, B. S., Gillig, J. R., Schwab, J. M. and Smith, J. L. (1996)

Structure of a dehydratase-isomerase from the bacterial pathway for biosynthesis of

unsaturated fatty acids: two catalytic activities in one active site, Structure 4, 253-

264.

112 Zhang, Y-M., Rao, M.S., Heath, R.J., Price, A.C., Olson, A.J., Rock, C.O. and

White, S.W. (2001) Identification and analysis of the acyl carrier protein (ACP)

docking site on β-ketoacyl-ACP synthase III, J Biol Chem. 276, 8231-8238.

113 Keatinge-Clay, A.T., Shelat, A. A., Savage, D. F., Tsai, S-C., Miercke, L. J.,

O'Connell, J. D., Khosla, C. and Stroud, R. M. (2003) Catalysis, Specificity, and

ACP Docking Site of Streptomyces coelicolor Malonyl-CoA:ACP Transacylase,

Structure 11, 147-154.

114 Zhang, L., Liu, W., Xiao, J., Hu, T., Chen, J., Chen, K., Jiang, H., and Shen, X.

(2007) Malonyl-CoA: acyl carrier protein transacylase from Helicobacter pylori:

Crystal structure and its interaction with acyl carrier protein, Protein Sci. 16, 1184-

1192.

115 Lai, J. R., Koglin, A and Walsh, C. T. (2006) Carrier Protein Structure and

Recognition in Polyketide and Nonribosomal Peptide Biosynthesis, Biochemistry

45, 14869-14879.

116 Parris, K. D., Lin, L., Tam, A., Mathew, R., Hixon, J., Stahl, M., Fritz, C. C.,

Seehra, J., and Somers, W. S. (2000) Crystal structures of substrate binding to


168

Bacillus subtilis holo-(acyl carrier protein) synthase reveal a novel trimeric

arrangement of molecules resulting in three active sites, Structure 8, 883–895.

117 Rafi, S., Novichenok, P., Kolappan, S., Zhang, X., Stratton, C. F., Rawat, R.,

Kisker, C., Simmerling, C. and Tonge, P. J. (2006) Structure of acyl carrier protein

bound to FabI, the FASII Enoyl Reductase from Escherichia coli, J Biol Chem. 281,

39285-39293.

118 Zhang, Y. M., Wu, B. N., Zheng, J. and Rock, C.O. (2003) Key residues responsible

for acyl carrier protein and β-ketoacyl-acyl carrier protein reductase (FabG)

interaction, J Biol Chem. 278, 52935-52943.

119 De Lay, N. R. and Cronan, J. E. (2007) In Vivo Functional Analyses of the Type II

Acyl Carrier Proteins of Fatty Acid Biosynthesis, J Biol Chem. 282, 20319-20328.

120 Arthur, C. J. (2004) PhD thesis, University of Bristol, UK.

121 Leibundgut, M., Jenni, S., Frick, C., Ban, N. (2007) Structural Basis for Substrate

Delivery by Acyl Carrier Protein in the Yeast Fatty Acid Synthase, Science 316,

288-290.

122 Westcott, J. (2001) PhD thesis, University of Bristol.

123 Burmeister Getz E., Xiao, M., Chakrabarty, T., Cooke, R. and Selvin, P. (1999) A

Comparison between the Sulfhydryl Reductants Tris(2-carboxyethyl)phosphine and

Dithiothreitol for Use in Protein Biochemistry, Anal Biochem. 273, 73-80.

124 Izumikawa, M., Cheng, Q. and Moore, B. S. (2006) Priming Type II Polyketide

Synthase via a Type II Nonribosomal Peptide Synthetase Mechanism, J Am Chem

Soc. 128, 1428-1429.

125 Hamada, M., Koike, K., Nakaula, Y., Hiraoka, T., Koike, M. and Hashimoto, T.

(1975) A kinetic study of the α-keto acid dehydrogenase complexes from pig heart

mitochondria, J Biochem 77, 1047-1056.

126 Bunik, V., Westpahl, A.H and de Kok, A. (2000) Kinetic properties of the 2-

oxoglutarate dehydrogenase complex from Azotobacter vinelandii evidence for the

formation of a precatalytic complex with 2-oxoglutarate, Eur J Biochemi. 267,

3583-3591.

127 Khandekar, S.S., Gentry, D.R., van Aller, G.S., Warren, P., Xiang, H., Silverman,

C., Doyle, M.L., Chambers, P.A., Konstantinidis, A.K., Brandt, M., Daines, R.A

and Lonsdale, J.T. (2001) Identification, substrate specificity, and inhibition of the


169

Streptococcus pneumoniae β-ketoacyl-acyl carrier protein synthase III (FabH), J

Biol Chem. 276, 30024-30030.

128 Molnos, J., Gardiner, R., Dale, G.E. and Lange, R. (2003) A continuous coupled

enzyme assay for bacterial malonyl-CoA:acyl carrier protein transacylase (FabD),

Anal Biochem. 319, 171-176.

129 Zha, W., Rubin-Pitel, S.B. and Zhao, H. (2006) Characterization of the substrate

specificity of PhlD, a type III polyketide synthase from Pseudomonas fluorescens, J

Biol Chem. 281, 32036-32047.

130 Liu, W., Han, C., Hu, L., Chen, K., Shen, X. and Jiang, H. (2006) Characterization

and inhibitor discovery of one novel malonyl-CoA: acyl carrier protein transacylase

(MCAT) from Helicobacter pylori, FEBS Lett. 580, 697-702 (2006).

131 Meadows, E.S. and Khosla, C. (2001) In vitro reconstitution and analysis of the

chain initiating enzymes of the R1128 polyketide synthase, Biochemistry 40, 14855-

14861.

132 Szafranska, A. E. PhD thesis, University of Bristol, 1999.

133 Kim, E-U., Cramer, K. D., Shreve, A. L. and Sherman, D. H. (1995) Heterologous

Expression of an Engineered Biosynthetic Pathway: Functional Dissection of Type

II Polyketide Synthase Components in Streptomyces Species, J Bacteriol. 177,

1202-1207.

134 Meurer, G. and Hutchinson C. R. (1995) Functional Analysis of Putative β-

Ketoacyl:Acyl Carrier Protein Synthase and Acyltransferase Active Site Motifs in a

Type II Polyketide Synthase of Streptomyces glaucescens, J Bacteriol. 177, 477-

481.

135 Roujeinikova, A., Baldock, C., Simon, W. J., Gilroy, J., Baker, P. J., Stuitje, A. R.,

Rice, D. W., Slabas, A. R., and Rafferty, J. B. (2002) X-ray crystallographic studies

on butyryl-ACP reveal flexibility of the structure around a putative acyl chain

binding site, Structure 10, 825-835.

136 Weber, T., Baumgartner, R., Renner, C., Marahiel, M. A. and Holak, T. A. (2000)

Solution structure of PCP, a Prototype for the Peptidyl Carrier Domains of Modular

Peptide Synthethases, Structure 8, 401-418.

137 Koglin, A., Mofid, M. R., Löhr, F., Schäfer, B., Rogov, V. V., Blum, M-M., Mittag,

T., Marahiel, M .A., Bernhard, A. and Dötsch, V. (2006) Conformational Switches


170

Modulate Protein Interactions in Peptide Antibiotic Synthetases, Science 312, 273-

276.

138 D’Agnolo, G., Rosenfeld, I. S. and Vagelos, P. R. (1975) Multiple forms of β-

ketoacyl-acyl carrier protein synthetase in Escherichia coli, J Biol Chem. 250, 5289-

5294.

139 Rosenfeld, I. S., D’Agnolo, G. and Vagelos, P. R. (1973) Synthesis of unsaturated

fatty acids and the lesion in fabB mutants, J Biol Chem. 248, 2452-2460.

140 Garwin J. L., Klages, A. L. and Cronan, J. E. Jr. (1980) Structural, Enzymatic, and

Genetic Studies of β-Ketoacyl-Acyl Carrier Protein Synthases I and II of

Escherichia coli, J Biol Chem. 255, 11949.

141 Jackowski, S. and Rock, C. O. (1987) Acetoacetyl-Acyl Carrier Protein Synthase, a

Potential Regulator of Fatty Acid Biosynthesis in Bacteria, J Biol Chem. 262, 7927-

7931.

142 Jackowski, S., Murphy, C. M., Cronan, J. E. Jr. and Rock, C. O. (1989)

Acetoacetyl-acyl Carrier Protein Synthase: a target for the antibiotic thiolactomycin,

J Biol Chem 264, 7624-7629..

143 Wattana-Amorn, P. (2007) PhD thesis, University of Bristol, UK.

144 Meurer, G., and Hutchinson, C. R. (1995) Daunorubicin type II polyketide synthase

enzymes DpsA and DpsB determine neither the choice of starter unit nor the

cyclization pattern of aromatic polyketides, J Am Chem Soc. 117, 5899–5900.

145 Bao, W., Sheldon, T. J., Wendt-Pienkowski, E. and Hutchinson, C. R. (1999) The

Streptomyces peucetius dpsC Gene Determines the Choice of Starter Unit in

Biosynthesis of the Daunorubicin Polyketide, J Bacteriol. 181, 4690-4695.

146 Bao, W., Sheldon, T. J. and Hutchinson, C. R. (1999) Purification and Properties of

the Streptomyces peucetius DpsC β-Ketoacyl:Acyl Carrier Protein Synthase III that

Specifies the Propionate-Starter Unit for Type II Polyketide Biosynthesis,


147 Hertweck, C., Luzhetskyy, A., Reberts, Y and Bechthold, A. (2007) Type II

polyketide synthases: gaining a deeper insight into enzymatic teamwork, Nat Prod

Rep. 24, 162-190.


171

148 Bibb, M. J., Sherman, D. H., Omura, S. and Hopwood, D. A. (1994) Cloning,

sequencing and deduced functions of a cluster of Streptomyces genes probably

encoding biosynthesis of the polyketide antibiotic frenolicin, Gene 142, 31-39.

149 Xu, Z., Schenk, A and Hertweck, C. (2007) Molecular Analysis for the Benastatin

Biosynthetic Pathway and Genetic Engineering of Altered Fatty Acid-Polyketide

Hybrids, J Am Chem Soc. 129, 6022-6030.

150 Marti, T., Hu, Z., Pohl, N. L., Shah, A. N., and Khosla, C. (2000) Cloning,

nucleotide sequence, and heterologous expression of the biosynthetic gene cluster

for R1128, a non-steroidal estrogen receptor antagonist. Insights into an unusual

priming mechanism, J Biol Chem. 275, 33443-33448.

151 Meadows, E. S and Khosla, C. (2001) In Vitro Reconstitution and Analysis of the

Chain Initiating Enzymes of the R1128 Polyketide Synthase, Biochemistry 40,

14855-14861.

152 Tang, Y., Koppisch, A. C and Khosla, C. (2004) The acyltransferase homologue

from the initiation module of the R1128 polyketide synthase is an acyl-ACP

thioesterase that edits acetyl primer units, Biochemistry 43, 9546-9555.

153 Tang, Y., Lee, H. Y., Tang, Y., Kim, C-Y., Mathews, I. and Khosla, C. (2006)

Structural and Functional Studies on SCO1815: A β-Ketoacyl-Acyl Carrier Protein

Reductase from Streptomyces coelicolor A3(2), Biochemistry 45, 14085-14093.

154 Sherman, D. H., Kim, E-S., Bibb, M. J. and Hopwood, D. A. (1992) Functional

Replacement of Genes for Individual Polyketide Synthase Components in

Streptomyces coelicolor A3(2) by Heterologous Genes from a Different Polyketide

Pathway, J Bacteriol. 174, 6184-6190.

155 Kresze, G-B., Steber, L., Oesterhelt, D. and Lynen, F. (1997) Reaction of yeast fatty

acid synthetase with iodoacetamide. 1. Kinetics of inactivation and extent of

carboxamidomethylation, Eur J Biochem. 79, 173-180.

156 Huang W., Jia J., Edwards P., Dehesh K., Schneider G. and Lindqvist Y. (1998)

Crystal structure of β-ketoacyl-acyl carrier protein synthase II from E. coli reveals

the molecular architecture of condensing enzymes. EMBO J. 17, 1183-1191.

157 Jez, J.M., Ferrer, J.L., Bowman, M.E., Dixon, R.A. and Noel, J.P. (2000) Dissection

of malonyl-coenzyme A decarboxylation from polyketide formation in the reaction

mechanism of a plant polyketide synthase, Biochemistry 39, 890-902.

158 Thwaite, J. S. (2003) PhD thesis, University of Cambridge, UK.


172

159 Katnar, S. S., Cleland, W. W. and Porter, J. W. (1975) Fatty Acid Synthetase: A

steady state kinetic analysis of the reaction catalyzed by the enzyme from

pigeon liver, J Biol Chem. 250, 2709-2717.

160 Ghayourmanesh, S. and Kumar, S. (1981) Synthesis of acetoacetyl-CoA by bovine

mammary fatty acid synthase, FEBS Lett. 132, 231-4.

161 Cox, B. G. and Hammes, G. G. (1983) Steady-state kinetic study of fatty acid

synthase from chicken liver, P Natl Acad Sci U. S. A 80, 4233-7.

162 Carlisle-Moore, L., Gordon, C. R., Machutta, C. A., Miller, W. T. and Tonge, P. T.

(2005) Substrate recognition by the human fatty acid synthase, J Biol Chem. 280,

42612-42618.

163 Wood, W. I., Peterson, D. O. and Bloch, K. (1977) Mycobacterium smegmatis

Fatty Acid Synthetase: A Mechanism Based On Steady State Rates And

Product Distributions, J Biol Chem. 252, 5745-5749.

164 Walker, T. A., Jonak, Z. L., Worsham, L. M. and Ernst-Fonberg M. L. (1981)

Kinetic studies of the fatty acid synthetase multienzyme complex from Euglena

gracilis variety bacillaris, Biochem J. 199, 383-392.

165 Jackowski, S., Murphys, C. M., Cronan, J. E. and Rock, C. O. (1989) Acetoacetyl-

acyl Carrier Protein Synthase: a target for the antibiotic thiolactomycin, J Biol

Chem. 264, 7624-7629.

166 Heath, R. J. and Rock, C. O. (1996) Inhibition of β-ketoacyl-acyl carrier protein

synthase III (FabH) by acyl-acyl carrier protein in Escherichia coli, J Biol Chem.

271, 10996-1000.

167 Garwin J. L., Klages, A. L. and Cronan, J. E. Jr. (1980) Structural, enzymatic, and

genetic studies of β-ketoacyl-acyl carrier protein synthases I and II of Escherichia

coli, J Biol Chem. 255, 11949-56.

168 He, X. and Reynolds, K. A. (2002) Purification, Characterization, and Identification

of Novel Inhibitors of the β-Ketoacyl-Acyl Carrier Protein Synthase III (FabH)

from Staphylococcus aureus, Antimicrob Agents Ch. 46, 1310-1318.

169 Schaeffer M. L., Agnihotri G., Volker C., Kallender H., Brennan P. J. and Lonsdale

J. T. (2001) Purification and biochemical characterization of the Mycobacterium

tuberculosis β-ketoacyl-acyl carrier protein synthases KasA and KasB, J Biol Chem.

270, 47029-47037.


173

170 Abbadi, A., Brummel, M., Schutt, B. S., Slabaugh, M. B., Schuch, R. and Spener, F.

(2000) Reaction mechanism of recombinant 3-oxoacyl-(acyl-carrier-protein)

synthase III from Cuphea wrightii embryo, a fatty acid synthase type II condensing

enzyme, Biochem J. 345, 153-160.

171 Funa, N., Awakawa, T. and Horinouchi, S. (2007) Pentaketide resorcylic acid

synthesis by type III polyketide synthase from Neurospora crassa, J Biol Chem.

online.

172 Jez, J. M., Ferrer, J.L., Bowman, M. E., Dixon, R. A. and Noel, J. P. (2000)

Dissection of malonyl-coenzyme A decarboxylation from polyketide formation in

the reaction mechanism of a plant polyketide synthase, Biochemistry 39, 890-902.

173 Wanibuchi, K., Zhang, P., Abe, T., Morita, H., Kohno, T., Chen, G., Noguchi, H.

and Abe, I. (2007) An acridone-producing novel multifunctional type III polyketide

synthase from Huperzia serrata. FEBS J. 274, 1073-1082.

174 Abe, I., Morita, H., Nomura, A. and Noguchi, H. (2000) Substrate specificity of

chalcone synthase: Enzymatic formation of unnatural aromatic polyketides from

synthetic cinnamoyl-CoA analogues, J Am Chem Soc. 122, 11242-11243.

175 Abe, I., Utsumi, Y., Oguro, S., Morita, H., Sano, Y. and Noguchi, H. (2005) A plant

type III polyketide synthase that produces pentaketide chromone, J Am Chem Soc.

127, 1362-1363.

176 Abe, I., Utsumi, Y., H., Sano, Y. and Noguchi, H. (2005) Engineered biosynthesis

of plant polyketides: chain length control in an octaketide-producing plant type III

polyketide synthase, J Am Chem Soc. 127, 12709-12716.

177 Funa, N., Ohnishi, Y., Fujii, I., Shibuya, M., Ebizuka, Y. and Horinouchi, S. (1999)

A new pathway for polyketide biosynthesis in microorganisms, Nature 400, 897-

899.

178 Funa, N., Ohnishi, Y., Ebizuka, Y. and Horinouchi, S. (2002) Properties and

substrate specificity of RppA, a chalcone synthase-related polyketide synthase in

Streptomyces griseus, J Biol Chem. 277, 4628-4635.

179 Li, S., Gruschow, S., Dordick, J. S. and Sherman, D. H. (2007) Molecular Analysis

of the Role of Tyrosine 224 in the active site of Streptomyces coelicolor RppA, a

Bacterial Type III Polyketide Synthase, J Biol Chem. 282, 12765-12772.


174

180 Zha, W., Rubin-Pitel, S B. and Zhao, H. (2006) Characterization of the Substrate

Specificity of PhID, a Type III Polyketide Synthase from Pseudomonas fluorescens,

J Biol Chem. 281, 32036-32047.

181 Gokhale, R. S., Tsuji, S. Y., Cane, D. E. and Khosla, C. (1999) Dissecting and

Exploiting Intermodular Communication in Polyketide Synthases, Science 284,

482-485.

182 Wu, N., Kudo, F., Cane, D. E and Khosla, C. (2000) Analysis of the Molecular

Recognition Features of Individual Modules Derived from the Erythromycin

Polyketide Synthase, J Am Chem Soc. 122, 4847-4852

183 Wu, N., Tsuji, S. Y., Cane, D. E and Khosla, C. (2001) Assessing the Balance

between Protein-Protein Interactions and Enzyme-Substrate Interactions in the

Chanelling of Intermediates between Polyketide Synthase Modules, J Am Chem

Soc. 123, 6465-6474.

184 Chen, A. Y., Schnarr, N. A., Kim, C-Y. Cane, D. C. and Khosla, C. (2006) Extender

Unit and Acyl Carrier Protein Specificity of Ketosynthase Domains of the 6-

Deoxyerythronolide B Synthase, J Am Chem Soc. 128, 3167-3074.

185 Winston, R. L. and Fitzgerald, M. C. (2000) Concentration and desalting of protein

samples for mass spectrometry analysis, Anal Biochem. 262, 83-85.

186 Lau, J., Cane, D. E. and Khosla, C. (2000) Dissecting the Role of Acyltransferase

Domains of Modular Polyketide Synthases in the Choice and Stereochemical Fate

of Extender Units, Biochemistry 39, 10514-10520.

187 Liou, G. F., Lau, J., Cane, D. E., Khosla, C. (2003) Quantitative analysis of loading

and extender acyltransferases of modular polyketide synthases, Biochemistry 42,

200-207.

188 Shen, B. and Hutchinson, C. R. (1996) Deciphering the mechanism for the assembly

of aromatic polyketides by a bacterial polyketide synthase, P Natl Acad Sci. U. S. A.

93, 6600-6604.

189 Dreier, J. and Khosla, C. (2000) Mechanistic Analysis of a Type II Polyketide

Synthase. Role of Conserved Residues in the β-Ketoacyl Synthase-Chain Length

Factor Heterodimer, Biochemistry 39, 2088-2095.

190 Witkowski, A, Joshi, A .K., Lindqvist, Y and Smith, S. (1999) Conversion of a β-

Ketoacyl Synthase to a Malonyl Decarboxylase by Replacement of the Active-Site

Cysteine with Glutamine, Biochemistry 38, 11643-11650.


175

191 Austin, M. B. and Noel, J. P. (2003) The chalcone synthase superfamily of Type III

polyketide synthases, Nat Prod Rep. 20, 79-110.

192 Modis, Y. and Wierenga, R. K. (1999) A biosynthetic thiolase in complex with a

reaction intermediate: the crystal structure provides new insights into the catalytic

mechanism, Structure 7, 1279-1290.

193 Evans, S. E. (2007) PhD thesis, University of Bristol, UK.

194 Keating, D. H., Carey, M. R. and Cronan, J. E. (1995) The Unmodified (Apo) Form

of E. coli Acyl Carrier Protein is a Potent Inhibitor of Cell Growth, J Bacteriol. 270,

22229-22235.

195 McGuire, K. A., Siggard-Andersen, M., Bangera, M. G., Olsem J. G. and von

Wettstein-Knowles, P. (2001) β-Ketoacyl-[Acyl Carrier Protein] Synthase I of

Escherichia coli: Aspects of the Condensation Mechanism Revealed by Analyses of

Mutations in the Active Site Pocket, Biochemistry 40, 9836-9845.

196 Bartel, P. L., Zhu, C-B., Lampel, J. S., Dosch, D. C., Connors, N. C., Strohl, W. R.,

Beale, J. M. and Floss, H. G. (1990) Biosynthesis of Anthraquinones by

Interspecies Cloning of Actinorhodin Biosynthesis Genes in Streptomycetes:

Clarification of Actinorhodin Gene Functions, J Bacteriol. 172, 4816-4826.

197 Zhang, X., He, A., Adefarati, J., Gallucci, S. P., Cole, J. M., Beale, P. J., Keller, H.

G., and Floss, H. (1990) Mutactin, a Novel Polyketide from Streptomyces

coelicolor. Structure and Biosynthetic Relationship to Actinorhodin, J Org Chem.

55, 1682-1684.

198 Fu, H., Hopwood, D. A. and Khosla, C. (1994) Engineered biosynthesis of novel

polyketides: evidence for temporal, but not regiospecific, control of cyclization of

an aromatic polyketide precursor, Chem Biol. 1, 205-210.

199 McDaniel, R., Ebert-Khosla, S., Fu, H., Hopwood, D. A. and Khosla, C. (1994)

Engineered biosynthesis of novel polyketides: Influence of a downstream enzyme

on the catalytic specificity of a minimal aromatic polyketide synthase, P Natl Acad

Sci U.S.A 91, 11542-11546.

200 McDaniel, R., Ebert-Khosla, S., Fu, H., Hopwood, D. A. and Khosla, C. (1993)

Engineered biosynthesis of novel polyketides, Science 262, 1546-1550.

201 Kunnari, T., Ylihonko, C., Hautala, A., Klika, K. D., Matntsatla P. and Hakala, J.

(1999) Incorrectly folded aromatic polyketides from polyketide reductase deficient

mutants, Bioorg Med Chem Lett. 9, 2639-2642.


176

202 Korman, T. P., Hill, J. A., Vu, T. N. and Tsai, S-C. (2004) Structural Analysis of

Actinorhodin Polyketide Ketoreductase: Cofactor Binding and Substrate

Specificity, Biochemistry 43, 14529-14538.

203 Korman, T. P., Tan, J. H., Wong, J., Luo, R. and Tsai, S-C. (2008) Inhibition

Kinetics and Emodin Cocrystal Structure of a Type II Polyketide Ketoreductase,


204 Kalaitzis, J. A. and Moore, B. S. (2004) Heterologous Biosynthesis of Truncated

Hexaketides Derived from the Actinorhodin Polyketide Synthase, J Nat Prod. 67,

1419-1422

205 Hertweck, C., Xiang, L., Kalaitzis, J. A., Cheng, Q., Palzer, M. and Moore, B. S.

(2004) Context-Dependent Behavior of the Enterocin Iterative Polyketide Synthase:

A New Model for Ketoreduction, Chem Biol. 11, 461–468.

206 Teartasin, W., Limpkin, C., Glod, F., Cox, R. J., Simpson, T. J., Crosby, J., Crump,

M. P. and Hadfield, A. T. (2004) Expression, purification, and preliminary X-ray

diffraction analysis of a ketoreductase from a Type II polyketide synthase, Acta

Crystallogr D60, 1137-1138.

207 Oppermann, U., Filling, C., Hult, M., Shafqat, N., Wu, X., Lindh, M., Shafqat, J.,

Nordling, E., Kallberg, Y., Persson, B, and Jorrnvall, H. (2003) Short-chain

dehydrogenases / reductases: the 2002 update, Chem-Biol Interact. 143, 247-253.

208 Zawada, R. J. and Khosla, C. (1999) Heterologous expression, purification,

reconstitution and kinetic analysis of an extended type II polyketide synthase, Chem

Biol. 6, 607-615.

209 Geoghegan, K. F., Dixon, H. B., Rosner, P. J., Hoth, L. R., Lanzetti, A. J.,

Borzilleri, K. A., Marr, E. S., Pezzullo, L. H., Martin, L. B., LeMotte, P. K.,

McColl, A. S., Kamath, A. V. and Stroh, J. G. (1999) Spontaneous α-N-6-

phosphogluconoylation of a "His tag" in Escherichia coli: the cause of extra mass of

258 or 178 Da in fusion proteins, Anal Biochem 267, 169-184.

210 Ma, S. M., Zhan, J., Xie, X., Watanabe, K., Tang, Y. and Zhang, W. (2008)

Redirecting the Cyclization Steps of Fungal Polyketide Synthase, J Am Chem Soc.

130, 38-39.

211 Khosla, C., McDaniel, R., Ebert-Khosla, S., Torres, R., Sherman, D. H., Bibb, M. J.,

and Hopwood, D. A (1993) Genetic construction and functional analysis of hybrid


177

polyketide synthases containing heterologous acyl carrier proteins, J Bacteriol. 175,

2197-2204.

212 Decker, H., Summers, R. G., and Hutchinson, C. R. (1994) Overproduction of the

acyl carrier protein component of a Type II polyketide synthase stimulates

production of tetracenomycin biosynthetic intermediates in Streptomyces

glaucescens, J Antibiot. 47, 54-63.


178

APPENDIX I: Michaelis-Menten plots for act mutant acyl carrier proteins, gris ACP and

dps ACP as a substrate for act KSα/KSβ


179

APPENDIX II: Sequence alignment of FAS and PKS ketosynthases


180


181

APPENDIX III. ESMS time-course study of self-malonylation of the reference act ACP

and an E47A ACP mutant. A: initial sample; B: 5 min; C: 15 min; D: 30 min; E: 120 min

A B

C D

E

Figure III.1. 50 µM ACP C17S ACP. Expected masses: 9441 (holo-ACP) and 9527

(malonyl-ACP).

holo

holo

holo holo

holo

malonyl

malonyl

malonyl

malonyl


182

A B

C D

Figure III.2. 25 µM C17S ACP. Expected masses: 9441 (holo-ACP) and 9527 (malonyl-

ACP).

malonyl

malonyl malonyl

holo

holo

holo

holo


183

A B

C D

E

Figure III.3. 50 µM E47A ACP. Expected masses: 9384 (holo-ACP) and 9470 (malonyl-

ACP).

holo

holo

holo

holo

holo

malonyl

malonyl

malonyl

malonyl


184

A B

C D

Figure III.4. 25 µM E47A ACP. Expected masses: 9384 (holo-ACP) and 9470 (malonyl-

ACP).

malonyl

malonyl

malonyl

holo

holo

holo

holo

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