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CHAPTER SIXTEEN Tailoring Enzymes Acting on Carrier Protein-Tethered Substrates in Natural Product Biosynthesis Shuangjun Lin*, Tingting Huang { , Ben Shen {,{,},1 *The State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, PR China { Department of Chemistry, The Scripps Research Institute, Jupiter, Florida, USA { Department of Molecular Therapeutics, The Scripps Research Institute, Jupiter, Florida, USA } Natural Products Library Initiative at TSRI, The Scripps Research Institute, Jupiter, Florida, USA 1 Corresponding author: e-mail address: [email protected] Contents 1. Introduction 322 2. Methods 331 2.1 In vitro characterization of SgcC3-catalyzed chlorination of (S)-b-tyrosyl-SgcC2 331 2.2 In vitro characterization of SgcC-catalyzed hydroxylation of (S)-b-3-chloro-tyrosinyl-SgcC2 336 2.3 Exploitation of SgcC2-tethered (S)-b-tyrosine analogues for structural diversification 338 3. Conclusion 339 Acknowledgment 340 References 340 Abstract Carrier proteins (CPs) are integral components of fatty acid synthases, polyketide synthases, and nonribosomal peptide synthetases and play critical roles in the biosyn- thesis of fatty acids, polyketides, and nonribosomal peptides. An emerging role CPs play in natural product biosynthesis involves tailoring enzymes that act on CP-tethered sub- strates. These enzymes provide a new opportunity to engineer natural product diversity by exploiting CPs to increase substrate promiscuity for the tailoring steps. This chapter describes protocols for in vitro biochemical characterization of SgcC3 and SgcC that cat- alyze chlorination and hydroxylation of SgcC2-tethered (S)-b-tyrosine and analogues in the biosynthesis of the enediyne chromophore of the chromoprotein C-1027. These protocols are applicable to mechanistic characterization and engineered exploitation of other tailoring enzymes that act on CP-tethered substrates in natural product Methods in Enzymology, Volume 516 # 2012 Elsevier Inc. ISSN 0076-6879 All rights reserved. http://dx.doi.org/10.1016/B978-0-12-394291-3.00008-3 321

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CHAPTER SIXTEEN

Tailoring Enzymes Acting onCarrier Protein-TetheredSubstrates in Natural ProductBiosynthesisShuangjun Lin*, Tingting Huang{, Ben Shen{,{,},1*The State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai JiaoTong University, Shanghai, PR China{Department of Chemistry, The Scripps Research Institute, Jupiter, Florida, USA{Department of Molecular Therapeutics, The Scripps Research Institute, Jupiter, Florida, USA}Natural Products Library Initiative at TSRI, The Scripps Research Institute, Jupiter, Florida, USA1Corresponding author: e-mail address: [email protected]

Contents

1.

MetISShttp

Introduction

hods in Enzymology, Volume 516 # 2012 Elsevier Inc.N 0076-6879 All rights reserved.://dx.doi.org/10.1016/B978-0-12-394291-3.00008-3

322

2. Methods 331

2.1

In vitro characterization of SgcC3-catalyzed chlorination of(S)-b-tyrosyl-SgcC2 331

2.2

In vitro characterization of SgcC-catalyzed hydroxylationof (S)-b-3-chloro-tyrosinyl-SgcC2 336

2.3

Exploitation of SgcC2-tethered (S)-b-tyrosine analogues for structuraldiversification 338

3.

Conclusion 339 Acknowledgment 340 References 340

Abstract

Carrier proteins (CPs) are integral components of fatty acid synthases, polyketidesynthases, and nonribosomal peptide synthetases and play critical roles in the biosyn-thesis of fatty acids, polyketides, and nonribosomal peptides. An emerging role CPs playin natural product biosynthesis involves tailoring enzymes that act on CP-tethered sub-strates. These enzymes provide a new opportunity to engineer natural product diversityby exploiting CPs to increase substrate promiscuity for the tailoring steps. This chapterdescribes protocols for in vitro biochemical characterization of SgcC3 and SgcC that cat-alyze chlorination and hydroxylation of SgcC2-tethered (S)-b-tyrosine and analogues inthe biosynthesis of the enediyne chromophore of the chromoprotein C-1027. Theseprotocols are applicable to mechanistic characterization and engineered exploitationof other tailoring enzymes that act on CP-tethered substrates in natural product

321

322 Shuangjun Lin et al.

biosynthesis and structural diversification. The ultimate goal is to use the in vitro findingsto guide in vivo engineering of designer natural products.

1. INTRODUCTION

Acyl carrier proteins (ACPs) and peptidyl carrier proteins (PCPs) are

small (�10 kDa) proteins, existing as either a discrete protein in a type II mul-

tienzyme complex or a distinct domain interspersed among the catalytic do-

mains of a type I multifunctional megasynthase (Marahiel & Essen, 2009;

Mercer & Burkart, 2007; Shen, 2000; Staunton & Weissman, 2001;

Weissman, 2009). While the overall amino acid sequence identity among

the carrier proteins (CPs) is modest, they are characterized by a highly

conserved signature motif of GxxSL/I. The serine residue in this motif is

the site for 40-phosphopantetheinylation, a posttranslational modification

catalyzed by 40-phosphopantetheinyl transferases (PPTases) (Lambalot et al.,

1996; Sanchez, Du, Edwards, Toney, & Shen, 2001). PPTases convert the

apo-CPs into the functional holo-CPs by installing the 20 A-long 40-phosphopantetheine prosthetic group with a free terminal thiol

(Fig. 16.1A). At this thiol, both substrates and the growing intermediates

are tethered as thioesters. While the 40-phosphopantetheinyl arm facilitates

the delivery of substrates into each of the active sites and channels the

growing intermediates between each of the elongation cycles, the CPs

provide necessary protein–protein recognition among the various

enzymatic partners.

CPs that carry short carboxylic acids or other acyl intermediates

are known as ACPs, which were first characterized from fatty acid syn-

thases (FASs) (Chan & Vogel, 2010; Gago, Diacovich, Arabolaza, Tsai, &

Gramajo, 2011; Mercer & Burkart, 2007). Type I FASs are multifunctional

proteins consisting of domains for individual activities, while type II

FASs are multienzyme complexes consisting of discrete, monofunctional

proteins. ACPs, either as a domain in type I FASs or a discrete protein in

type II FASs, play a pivotal role in fatty acid biosynthesis by tethering the

starter and extender units for condensation and by channeling the growing

acyl intermediates for complete b-ketoreduction (i.e., b-ketoreduction,dehydration, and enoylreduction) during each cycle of chain elongation

to afford the fully reduced fatty acid as the final product (Fig. 16.1B).

PPTaseOH

apo-ACPapo-PCP holo-ACP

holo-PCP

CoA ADP

ACP

SH SO

OO

OO

OR

S

R S-Enz

ACP ACP ACP ACP

Fatty acids

S

R Rn

OS

O

b-Ketoreduction(complete)ElongationAT

O OHNH

NH

PO O

OO

4�-PhosphopantetheineA

B

C

D

E

OSH

SH

ACP

PCP

A/P-CP A/P-CPTailoring enzymes

CyclizationHalogenationMethylationOxidationReduction

(see Table 16.1 for examples)

Natural products(see Fig. 16.2 for examples)

AT/A

SH SO

A/P-CP

SO

Rs:

Rs

Groups introducedby tailoring enzymes

PCP PCP PCP

S S

O O

O

OPCP

S

A

SH SO

O O

R1

R2

R1

R2

R1

NH2

NH2 H2N

NH

SH SO

OO

OO

OR

S

R S-Enz

ACP ACP ACP ACP

S

R

O

OH

NH2

Rn+1

Rn

nNH

HN

S

O OPolyketides

Peptides

OH

R

b-Ketoreduction(selective)Elongation

Elongation

AT

Figure 16.1 Carrier proteins and their roles in fatty acid, polyketide, and nonribosomalpeptide biosynthesis: (A) posttranslational modification of an apo-ACP or apo-PCP into aholo-ACP or holo-PCP by a PPTase; (B) ACP-mediated substrate activation and interme-diate channeling in fatty acid biosynthesis; (C) ACP-mediated substrate activation andintermediate channeling in polyketide biosynthesis; (D) PCP-mediated substrate activa-tion and intermediate channeling in nonribosomal peptide biosynthesis; and (E) tailor-ing enzymes acting on ACP- or PCP-tethered substrates in natural product biosynthesis.See Table 16.1 for specific tailoring enzymes, ACP- or PCP-tethered substrates and theircorresponding products, and the types of modification and Fig. 16.2 for structures ofnatural products with moieties modified by tailoring enzymes highlighted in gray.A, adenylation enzyme; ACP, acyl carrier protein; AT, acyltransferase; PCP, peptidylcarrier protein; PPTase, 40-phosphopantetheinyl transferase.

323Tailoring Enzymes Acting on Carry Protein-Tethered Substrates

324 Shuangjun Lin et al.

ACPswere subsequently characterized frompolyketide synthases (PKSs),

which catalyze the biosynthesis of polyketides, a large family of natural prod-

uctswithprofoundbiological activities (Mercer&Burkart, 2007; Shen, 2000;

Staunton&Weissman, 2001;Weissman, 2009). Following the convention of

FASs, PKSs have also been classified into types I and II according to their

enzyme architectures (Shen, 2003). Thus, similar to FASs, ACPs in type I

PKSs are domains, ACPs in type II PKSs are discrete proteins, and

regardless of their architectural difference, both ACP domains and proteins

tether the acyl CoA substrates for condensation and channel the growing

acyl intermediates during each cycle of chain elongation. However, in

contrast to FASs, the b-ketone groups of the ACP-tethered growing acyl

intermediates in PKSs can undergo no, partial, or full reduction,

depending on the given cycle of elongation, thereby providing a

mechanistic basis to account for the vast structural diversity of polyketide

natural products (Fig. 16.1C).

CPs from nonribosomal peptide synthetases (NRPSs) are known as

PCPs, carrying amino acids or peptidyl intermediates. NRPSs catalyze

the biosynthesis of nonribosomal peptides, another major family of natural

products including many clinically important drugs (Marahiel & Essen,

2009; Mercer & Burkart, 2007). Although PKSs and NRPSs catalyze the

biosynthesis of two distinct classes of natural products from two different

pools of substrates, they apparently use a very similar molecular logic for

substrate activation and intermediate channeling. While the type I and II

nomenclature for FASs and PKSs has not been widely accepted to classify

NRPSs, both multifunctional NRPSs with distinct domains and discrete

NRPSs with largely monofunctions are known. In a mechanism

analogous to FASs and PKSs, NRPSs use PCPs to tether the amino acid

substrates for condensation and channel the growing peptidyl

intermediates during each cycle of chain elongation (Fig. 16.1D). These

striking structural and mechanistic similarities between PKSs and NRPSs

have inspired the discovery and characterization of natural NRPS–PKS

megasynthases for the biosynthesis of hybrid peptide–polyketide natural

products and the construction of engineered hybrid NRPS–PKS systems

to further expand the size and diversity of natural product libraries

(Du et al., 2001; Fischbach & Walsh, 2006, 2010).

CP-dependent PKSs and NRPSs catalyze the assembly of a myriad of

polyketide, peptide, and hybrid polyketide–peptide backbones from a vast

array of short carboxylic acids and amino acids. The nascent scaffolds are

often heavily modified by the coordinated action of specialized enzymes,

325Tailoring Enzymes Acting on Carry Protein-Tethered Substrates

known as tailoring enzymes, to further imbue structural and functional

diversity. While tailoring enzymes that act during chain elongation, that

is, with the growing intermediates still tethered to specific ACPs or PCPs,

are known, most tailoring enzymes act on the peptide, polyketide, or hybrid

peptide–polyketide intermediates after they are released from the PKS or

NRPS megasynthases as free substrates (Fischbach & Walsh, 2010; Walsh

et al., 2001).

A subset of tailoring enzymes is emerging that specifically act on CP-

tethered substrates; the corresponding free substrates are not recognized.

This strategy is most commonly associated with biosynthesis of unusual

building blocks incorporated into many polyketide and nonribosomal pep-

tide natural products. Modifications catalyzed by tailoring enzymes acting

on both ACP- and PCP-tethered substrates are known, including cycliza-

tion, halogenation, methylation, oxidation (dehydrogenation, epoxidation,

and hydroxylation), and reduction (Fig. 16.1E). Table 16.1 summarizes the

tailoring enzymes known to date that have been biochemically characterized

and act on CP-tethered substrates in natural product biosynthesis (Fig. 16.2).

Tailoring enzymes that act on CP-tethered substrates therefore represent a

new molecular logic for natural product biosynthesis. The tethering of pre-

cursors to CPs ensures that the resultant building blocks will be sequestered

from endogenous metabolite pools and efficiently incorporated into the final

natural products.

The enediyne chromophore of the C-1027 chromoprotein, one of the

most potent antitumor antibiotics known to date, features a highly modified

b-amino acid moiety (Fig. 16.3; Van Lanen & Shen, 2008). The gene cluster

for C-1027 biosynthesis was cloned and sequenced from Streptomyces

globisporus (Liu, Christenson, Standage, & Shen, 2002). Bioinformatics anal-

ysis of the genes within the C-1027 biosynthetic gene cluster predicted, and

biochemical characterizations subsequently confirmed, that the biosynthesis

of the b-amino acid moiety from the a-tyrosine precursor involved tailoringenzymes that act on PCP-tethered substrates (Van Lanen et al., 2005). Thus,

a-tyrosine is first converted by the SgcC4 aminomutase to (S)-b-tyrosine(Christenson, Liu, Toney, & Shen, 2003; Christenson, Wu, Spies, Shen, &

Toney, 2003), which is then tethered by the SgcC1 adenylation enzyme to

the SgcC2 PCP (Van Lanen, Lin, Dorrestein, Kelleher, & Shen, 2006).

Sequential chlorination and hydroxylation of the SgcC2-tethered (S)-b-tyrosine by the SgcC3 halogenase (Lin et al., 2007) and SgcC

monooxygenase (Lin et al., 2008), respectively, affords the fully modified

b-tyrosine building block, which, still tethered to the SgcC2 PCP, is

Table 16.1 Tailoring enzymes acting on carrier protein-tethered substrates that have been biochemically characterized from natural productbiosynthetic pathways.

Natural productsaTailoringenzyme

Carrierprotein Type of reaction Substrates Products Reference

Armentomycin CytC3 CytC2 Chlorination L-aminobutanoic

acid

g,g-Dichloro-L-

aminobutanoic acid

Ueki et al. (2006)

Barbamide BarB1

BarB2

PCPBarA Chlorination L-leucine d-trichloro-L-leucine Galonic,

Vaillancourt, and

Walsh (2006)

Flatt et al. (2006)

C-1027 SgcC3 SgcC2 Chlorination (S)-b-tyrosine (S)-3-chloro-b-tyrosine Lin, Van Lanen, and

Shen (2007)

C-1027 SgcC SgcC2 Hydroxylation (S)-3-chloro-b-tyrosine

(S)-3-chloro-5-hydroxy-b-tyrosine

Lin, Van Lanen, and

Shen (2008)

CDA HxcO ACP Dehydrogenation hexanoic acid hex-2-enoic acid Kopp, Linne,

Oberthur, and

Marahiel (2008)

CDA HcmO ACP Oxidation hex-2-enoic acid 2,3-Epoxyhexanoic acid Kopp et al. (2008)

Chloramphenicol CmlA PCPCmlP Hydroxylation L-p-

aminophenylalanine

b-Hydroxy-L-p-

aminophenylalanine

Makris, Chakrabarti,

Munck, and

Lipscomb (2010)

Chlorobiocin CloN3 CloN5 Dehydrogenation L-proline Pyrrole-2-carboxylic acid Garneau-Tsodikova,

Dorrestein, Kelleher,

and Walsh (2005)

Coronatine CmaB CmaD Chlorination L-allo-isoleucine Chloro-L-allo-isoleucine Vaillancourt, Yeh,

Vosburg, O’Connor,

and Walsh (2005)

Coronatine CmaC CmaD Cyclization g-Chloro-L-alloisoleucine

S,2S)-1-amino-2-

hylcyclopropanecarboxylic

id

Vaillancourt, et al.

(2005)

Dapdiamide DdaC PCPDpaD Epoxidation Nb-fumaramoyl

2,3-

diaminopropion

b-epoxysuccinamoyl-L-

3-diaminopropionate

Hollenhorst et al.

(2010)

FK506 TcsC ACPTcsA Reduction/

carboxylation

E-pent-2-enoic

acid

Propylmalonic acid Mo et al. (2011)

Kutzneride KtzD KtzC Chlorination L-isoleucine Chloro-L-isoleucine Neumann andWalsh

(2008)

Kutzneride KtzA KtzC Cyclization g-Chloro-L-isoleucine

S,2R)-1-amino-2-

hylcyclopropanecarboxylic

id

Neumann andWalsh

(2008)

Kutzneride KthP KtzC Chlorination Piperazate S, 5S)-5-chloropiperazate Jiang et al. (2011)

Kutzneride KtzO PCPKtzH Hydroxylation L-glutamic acid threo-b-hydroxy-glutamic

id

Strieker, Nolan,

Walsh, and Marahiel

(2009)

Continued

g-

- (1

et

ac

-L-

ate

N

2,

2-

g-

(1

et

ac

(3

L-

ac

Table 16.1 Tailoring enzymes acting on carrier protein-tethered substrates that have been biochemically characterized from naturalproduct biosynthetic pathways.—cont'd

Natural productsTailoringenzyme

Carrierprotein Type of reaction Substrates Products Reference

Kutzneride KtzP PCPKtzH Hydroxylation L-glutamic acid L-erythro-b-hydroxy-glutamic acid

Strieker et al. (2009)

Nikkomycin NikQ PCPNikP1 Hydroxylation Histidine b-Hydroxy-histidine Chen, Hubbard,

O’Connor, and

Walsh (2002)

Novobiocin NovI PCPNovH Hydroxylation L-tyrosine b-Hydroxy-L-tyrosine Chen and Walsh

(2001)

Novobiocin NovJ/K PCPNovH Oxidation b-OH-L-tyrosine b-Ketotyrosine Pacholec, Hillson,

and Walsh (2005)

Pacidamycin PacV PCPPacP Methylation L-2,3-

diaminobutyrate

L-3-N-methyl-2,3-

diamniobutyrate

Zhang et al. (2011)

Pyochelin PchG PCPPchF Reduction Hydroxyphenyl-

bisthiazolinic acid

Des-N-methyl-pyochelinic

acid

Reimmann et al.

(2001)

Pyoluteorin PltA PltL Chlorination Pyrrole-2-

carboxylic acid

4,5-Dichloropyrrole-2-

carboxylic acid

Dorrestein, Yeh,

Garneau-Tsodikova,

Kelleher, and Walsh

(2005)

Pyoluteorin PltE PltL Dehydrogenation L-proline Pyrrole-2-carboxylic acid Thomas, Burkart,

and Walsh (2002)

Sibiromycin SibG PCPSibE Hydroxylation 3-hydroxy-4-

methylanthranilic

acid

3,5-dihydroxy-4-

methylanthranilic acid.

Giessen, Kraas, and

Marahiel (2011)

Syringomycin E SyrB2 PCPSyrB1 Chlorination L-threonine g-Chloro-L-threonine Vaillancourt, Yin,

and Walsh (2005),

Blasiak, Vaillancourt,

Walsh, and Drennan

(2006)

Syringomycin E SyrP PCP8SyrE Hydroxylation L-aspartic acid L-threo-b-hydroxy-asparticacid

Singh, Fortin,

Koglin, and Walsh

(2008)

Undecylprodigiosin RedW ORF9 Dehydrogenation L-proline pyrrole-2-carboxylic acid Thomas et al. (2002)

Vancomycin OxyD PCPBpsD Hydroxylation (R)-tyrosine (R)-b-hydroxy-tyrosine Cryle, Meinhart, and

Schlichting (2010)

aSee Fig. 16.2 for structures of the natural products with moieties (highlighted in gray) that were modified by the tailoring enzymes acting on carrier protein-tetheredsubstrates.

Cl

Cl

O

Armentomycin(dichloroaminobutanoic acid)

Barbamide

N SO

N OMe

O

O

O O

O

O

O

O

O O

OO O

OOH

H

H

O

O

OO

O

O

OHO

NO2

HO

HO

HO

OH

HO

O

O

OO O

O

OO

HFK506

N

O

ClHO

HN

N NH

OH

HO

H2N

HN

HN

O

O

O

O O R

Nikkomycin (I, R = Glu)Nikkomycin (X, R = OH)

N

OH

OHH

N

NHCl

N NH OH

HN

CO2H

3

HO

Kutzneride 2 (3S)Kutzneride 8 (3R)

Pacidamycin 1

Pyochelin

Pyoluteoin

OOO

O

OO

O

OO

Cl

O

ChloramphenicolChlorobiocin (R = Cl)Novobiocin (R = CH

3)

Dapdiiamide ECoronatine

Cl

Cl R

OON O

OH

OH

NH2

CO2H

H2N

NH2

C-1027

ClOH

OH

OH

NH

O

O

O

O

OO

O

O

O

OCO

2H

HO2CHO

2C

CO2H

CDA

H2NOC

OO

OO

OHNH

NH NHOH

HN

OHO

NH

NH

HN

NH

HN

NH

NH N

HNH

HN

HN

HO

OH

HO Sibiromycin

Undecylprodigiosin

Vancomycin

Syringomycin ECl

O

O

O

OO

OO

O

OOOOH

OH

OH

NH

NH

NH

NH

NH

NH

HN

HN

HN

HN

HN

HN

OMe

C11H

23

H19C

9

HO2C NH

2

NH

NH2

H2N

NH2

HO

HO

HO

OHOH

OH

OH

HN

HO

HO2C

OH

OH

Cl

Cl

O

O O

OO

O

O O

O

OHN

O

OO

NH2

HN

HN

HN

HO

HO

HN

NH N

HN

N

N

O

O

O

H

OO

OO

O

O

O

OO

OHOH S

N N

S

OH

OH

O

Cl

Cl

OH

NH2

N

N

HN

HN

HN

HN

HN

NH

NH

NH

HN

NH

NH

NH

CCl3NH

2

OH

Figure 16.2 Structures of natural productswhose biosynthetic pathways feature tailoringenzymes that have been biochemically characterized to act on carrier protein-tetheredsubstrates. Moieties resulted from tailoring enzymes acting on carrier protein-tetheredsubstrates are highlighted in gray. See Table 16.1 for specific tailoring enzymes, ACP- orPCP-tethered substrates and their corresponding products, and the types of modification.

330 Shuangjun Lin et al.

incorporated directly into the C-1027 enediyne chromophore by the SgcC5

condensation enzyme (Lin, Huang, Horsman, Huang, Guo, & Shen, 2012;

Lin, Van Lanen, & Shen, 2009; Fig. 16.3).

In this chapter, we describe protocols for in vitro biochemical character-

ization of SgcC3 and SgcC that catalyze chlorination and hydroxylation of

SgcC2-tethered (S)-b-tyrosine and analogues. They include: (i) preparation

H

SgcC2

SgcC3

SgcC1

SgcC4

(S)-b-Tyr

OH

S

OH

2N

H

OH

O

O

ÅH

3N

L-Tyr

H

OH

O

O

ÅH

3N

H

SgcC2

SgcC

OH

S

O

Cl

H2N

H

SgcC2

SgcC5

Enediyne coreBenzoxazolinate

Deoxy aminosugarOH

OH

S

O

O

O

O O

O

OO

OON

OH

C-1027 (R1 = OH, R

2 = Cl)

20-Deschloro-C-1027 (R1 = OH, R

2 = H)

22-Deshydroxy-C-1027 (R1 = H, R

2 = Cl)

20-Deschloro-22-deshydroxy-C-1027 (R1 = R

2 = H)

OH

NH

O

Cl

H2N

NH2

R1

R2

Figure 16.3 Biosynthesis of the (S)-3-chloro-5-hydroxy-b-tyrosine moiety of C-1027 andengineered biosynthesis of C-1027 analogues. (S)-b-Tyrosine was first activated andtethered to the SgcC2 PCP by the SgcC1 adenylation enzyme. (S)-b-Tyrosyl-SgcC2was sequentially chlorinated by SgcC3 and hydroxylated by SgcC to afford (S)-3-chloro-5-hydroxy-b-tyrosyl-SgcC2, which was directly incorporated into C-1027 bySgcC5. Manipulation of SgcC3 or SgcC in C-1027 biosynthesis resulted in the productionof three C-1027 analogues, 20-deschloro-C-1027, 22-deshydroxy-C-1027, and20-deschloro-22-deshydroxy-C-1027.

331Tailoring Enzymes Acting on Carry Protein-Tethered Substrates

of the holo-SgcC2 PCP; (ii) preparation of SgcC2-tethered (S)-b-tyrosinesubstrates; (iii) SgcC3-catalyzed chlorination of (S)-b-tyrosyl-SgcC2;(iv) SgcC-catalyzed hydroxylation of (S)-3-chloro-b-tyrosyl-SgcC2; and(v) exploitation of SgcC2-tethered (S)-b-tyrosine substrates for structural

diversification.

2. METHODS

2.1. In vitro characterization of SgcC3-catalyzedchlorination of (S)-b-tyrosyl-SgcC2

SgcC3 is a FAD-dependent halogenase, acting only on SgcC2-tethered sub-

strates and accepting both (S)- and (R)-b-tyrosyl-SgcC2. SgcC3 catalyzes

preferentially chlorination but also bromination, and it does not catalyze

fluorination or iodination. SgcC3 requires Cl� (or Br�), O2, and reduced

FAD. The latter can be supplied by the C-1027 pathway-specific flavin

reductase SgcE6 orEscherichia coli flavin reductase Fre from FAD andNADH

(Fig. 16.4B; Lin et al., 2007).

2.1.1 Expression in E. coli and overproduction and purification ofapo-SgcC2

1. Most ACPs or PCPs of Streptomyces origin, upon expression in E. coli, are

overproduced in apo-form. Follow the protocols provided in Methods

Enzymology, volume 459 (Cheng, Coughlin, Lim, & Shen, 2009;

SgcC2 SgcC2

SgcC2 SgcC2

SgcC3

SgcE6

HO-X

FAD-OH

FAD FADH2

NADH NAD

FAD-OOH

OH

H2O

X

SgcC1

SgcC2

H3N H

2N

H2O

H2NH

O

S KOH

X

O2

H HO

O O

OH

S

OH

SH(S)-b-Tyr

ATP+

PPi+

AMP

ÅH

3NH O

O

OHX

(S)-3-chloro-b-Tyr (X = Cl)(S)-3-bromo-b-Tyr (X = Br)

Å

SgcC2 SgcC2

SgcC

SgcE6

FAD-OOH FAD-OH

FADH2

NAD NADH

FAD

OHOH

H2O

SgcC1

SgcC2

H3N H

2N H

2NH

O

S KOH

X

O2

H HO

O

X X

O

OH

S

OH

SH(S)-b-Tyr

ATP+

PPi+

AMP

ÅH

3NH O

O

OHOHX

(S)-3-hydroxy-b-Tyr (X = H)(S)-3-fluoro-5-hydroxy-b-Tyr (X = F)(S)-3-chloro-5-hydroxy-b-Tyr (X = Cl)(S)-3-bromo-5-hydroxy-b-Tyr (X = Br)(S)-3-iodo-5-hydroxy-b-Tyr (X = I)(S)-3-methyl-5-hydroxy-b-Tyr (X = CH

3)

Å

OH SH

apo-ACP

A

B

C

holo-ACP

Svp

CoA ADP

Figure 16.4 In vitro characterization of SgcC3 as a FAD-dependent halogenase andSgcC as a FAD-dependent hydroxylase that act on SgcC2-tethered (S)-b-tyrosine andanalogues: (A) Svp PPTase-catalyzed in vitro conversion of apo-SgcC2 into holo-SgcC2;(B) SgcC1-catalyzed preparation of (S)-b-tyrosyl-SgcC2, SgcC3-catalyzed chlorination orbromination of (S)-b-tyrosyl-SgcC2, and hydrolytic release from SgcC2 of the haloge-nated products (S)-3-chloro-b-tyrosine and (S)-3-bromo-b-tyrosine; and (C) SgcC1-catalyzed preparation of SgcC2-tethered (S)-b-tyrosine and analogues, SgcC-catalyzedhydroxylation of SgcC2-tethered (S)-b-tyrosine and analogues, and hydrolytic releasefrom SgcC2 of the hydroxylated products (S)-3-hydroxy-b-tyrosine, (S)-3-fluoro-5-hydroxy-b-tyrosine, (S)-3-chloro-5-hydroxy-b-tyrosine, (S)-3-bromo-5-hydroxy-b-tyro-sine, (S)-3-iodo-5-hydroxy-b-tyrosine, and (S)-3-methyl-5-hydroxy-b-tyrosine.

332 Shuangjun Lin et al.

Horsman, Van Lanen, & Shen, 2009; Jiang, Rajski, & Shen, 2009) to

express sgcC2 in E. coli BL21 (DE3) and to purify the overproduced

apo-SgcC2 as an N-terminal His6-tagged fusion protein.

2. Dialyze the purified SgcC2 into 50 mM Tris–HCl (pH 7.5), containing

50 mMNaCl and 1 mM dithiothreitol (DTT), and concentrate using an

Amicon Ultra-4 (3K, GE Healthcare, Piscataway, NJ).

3. Check the purity of the isolated protein by SDS-PAGE on a 15% gel

(Fig. 16.5A), determine the concentration by Bradford assay (Bio-

Rad, Hercules, CA), and store in 40% glycerol at �20 �C until use.

SgcE

6Sg

cC3

SgcC

2

SgcC

1

SgcC

MW

Std

s

MW

Std

s

MW

Std

s

kD

97

66

45

31

21

14

mAU at 280 nmB C D

A

15.0 20.0 18.0 20.0

Time (min) Time (min) Time (min)

22.0 24.0 15.0 20.0 25.0

I

II

III

I

II

III

I

II

III

mAU at 280 nm mAU at 280 nm

Figure 16.5 Representative data from in vitro characterization of SgcC3 and SgcC withSgcC2-tethered (S)-b-tyrosine and analogues as substrates. (A) SDS-PAGE analysis ofSgcC2, SgcC3, and SgcE6 on a 15% gel and SgcC1 and SgcC on a 12% gel. (B) HPLCchromatograms of SgcC3-catalyzed chlorination of (S)-b-tyrosyl-SgcC2: (I) authentic(S)-b-tyrosine standard (●), (II) assay solution, and (III) authentic (S)-3-chloro-b-tyrosinestandard (ç). (C) HPLC chromatograms of SgcC-catalyzed hydroxylation of (S)-b-tyrosyl-SgcC2: (I) authentic (S)-3-chloro-b-tyrosine standard (ç), (II) assay solution, (III) authentic(S)-3-chloro-5-hydroxy-b-tyrosine standard (r), and 4,5-dihydroxy-1,2-dithiane (*)presented in the assay. (D) HPLC chromatograms of SgcC3-catalyzed bromination of(S)-b-tyrosyl-SgcC2: (I) authentic (S)-b-tyrosine standard (●), (II) assay solution, and(III) authentic (S)-3-bromo-b-tyrosine standard (◊).

333Tailoring Enzymes Acting on Carry Protein-Tethered Substrates

2.1.2 In vitro preparation of holo-SgcC2 by Svp and of(S)-b-tyrosyl-SgcC2 by SgcC1

1. Follow the protocols provided inMethodsEnzymology, volume 459 (Cheng

et al., 2009; Horsman et al., 2009; Jiang et al., 2009) to convert apo-SgcC2

into holo-SgcC2 using the Svp PPTase (Sanchez et al., 2001; Fig. 16.4A).

Mix 0.8 mL of solution containing 160 mM apo-SgcC2, 0.8 mM

CoA, 12.5 mM MgCl2, and 2 mM tris(2-carboxyethyl)phosphine

hydrochloride (TCEP) in 100 mM Tris–HCl (pH 7.5), initiate the

reaction by adding 5 mM Svp, and incubate at 25 �C for 45 min.

334 Shuangjun Lin et al.

2. Express sgcC1 in E. coli BL21 (DE3) and purify the overproduced SgcC1

adenylation enzyme as an N-terminal His6-tagged fusion protein

according to the literature procedure (Fig. 16.5A; Van Lanen et al.,

2006); use SgcC1 to catalyze the tethering of (S)-b-tyrosine to

holo-SgcC2 (Fig. 16.4B). Add 0.8 mL of solution, containing 4 mM

(S)-b-tyrosine, 8 mM ATP, 2 mM TCEP, and 12.5 mM MgCl2, to

the holo-SgcC2 solution from step 1. Initiate the reaction by adding

2 mM SgcC1, and incubate at 25 �C for 1 h.

3. Purify (S)-b-tyrosyl-SgcC2 by ion exchange chromatography on a 5-mL

HiTrap Q column (GE Healthcare). Preequilibrate the column with

50 mM Bis–Tris–HCl (pH 7.0), load the reaction mixture from step 2

to the column, and wash it with five column volumes of the same buffer.

Elute the column with a linear gradient from 0% to 100% 1 M NaCl in

50 mM Bis–Tris–HCl (pH 7.0), in 25 column volumes at a flow rate of

3 mL/min. (S)-b-Tyrosinyl-SgcC2 is typically eluted between 0.35 and

0.4M NaCl.

4. Desalt b-tyrosyl-S-SgcC2 from step 3 using a Superose 12 column (GE

Healthcare) in 20 mM sodium phosphate, pH 7.0, and concentrate using

an Amicon Ultra-4 (3K, GE Healthcare) prior to use in SgcC3 assay.

2.1.3 Expression in E. coli and overproduction and purification of SgcC31. Prepare PCR primers for amplification of sgcC3 from cosmid pBS1005

(Liu et al., 2002), clone the PCR product into the pET-30Xa/LIC vector

(Novagen, Madison, WI) using a ligation-independent cloning procedure

to yield the expression plasmid pBS1041, and sequence the construct to

confirm PCR fidelity. With this construct, SgcC3 will be overproduced

as an N-terminal His6-tagged fusion protein (Lin et al., 2007).

2. Introduce pBS1041 into E. coli BL21 (DE3) by transformation, and select

transformants on LB agar plates containing 50 mg/mL kanamycin.

3. Pick a single colony to grow in 3 mL of LB containing 50 mg/mL kana-

mycin overnight at 37 �C, and transfer 0.5 mL into 50 mL of LB con-

taining 50 mg/mL kanamycin to grow again overnight at 37 �C to

prepare the seed culture. Inoculate 500 mL of LB containing 50 mg/mL kanamycin with 5 mL of the seed culture, and incubate at 18 �Cuntil

it reaches an OD600 of 0.6.

4. Induce sgcC3 expression by adding IPTG to 0.1 mM and continue incu-

bation at 18 �C for 15–20 h.

5. Harvest the cells by centrifugation at 4 �C, resuspend the cells in buffer A(100 mM sodium phosphate, pH 7.5, 300 mM NaCl) supplemented

335Tailoring Enzymes Acting on Carry Protein-Tethered Substrates

with a complete protease inhibitor tablet, EDTA-free (Roche Applied

Science, Indianapolis, IN), lyse the cells by sonication (4�30 s pulse

cycle), and centrifuge the lysate at 4 �C and 15,000 rpm for 30 min to

collect the clear supernatant.

6. Load the supernatant to a preequilibrated Ni-NTA agarose column

(Qiagen, Valencia, CA) with buffer B (buffer A plus 10% glycerol), and

wash the column sequentially with five column volumes of buffer B

and five column volumes of buffer B containing 20 mM imidazole. Elute

the column with five column volumes of buffer B containing 250 mM

imidazole, and pool fractions containing SgcC3.

7. Desalt the purified SgcC3 using a PD-10 column (GE Healthcare) into

50 mM Tris–HCl (pH 7.5), containing 50 mM NaCl and 1 mM DTT,

and concentrate using an Amicon Ultra-4 (10K, GE Healthcare).

8. Check the purity of the isolated protein by SDS-PAGE on a 15% gel

(Fig. 16.5A), determine the concentration by Bradford assay (Bio-Rad),

and store in 40% glycerol at �20 �C until use.

2.1.4 Expression in E. coli and overproduction and purification of SgcE61. Prepare PCR primers for amplification of sgcE6 from cosmid pBS1006

(Liu et al., 2002) and clone the PCR product into the pET-30Xa/LIC

vector (Novagen) using a ligation-independent cloning procedure to

yield the expression plasmid pBS1042, in which SgcE6 will be over-

produced as an N-terminal His6-tagged fusion protein. Sequence the

construct to confirm PCR fidelity.

2. Follow steps 2–8, Section 2.1.3, to afford pure SgcE6 (Fig. 16.5A), and

store in 40% glycerol at �20 �C until use.

2.1.5 In vitro assay of SgcC3-catalyzed chlorination of(S)-b-tyrosyl-SgcC2

1. Set up the SgcC3-catalyzed halogenation of (S)-b-tyrosinyl-SgcC2 in

200 mL of reaction solution, containing 50 mM (S)-b-tyrosyl-SgcC2,5 mM NADH, 0.10 mM FAD, 100 mM NaCl, 1 mM TCEP, and 5 mMSgcE6 in 50 mM sodium phosphate buffer (pH 6.0), at 37 �C (Fig. 16.4B).

2. Initiate the reaction by adding 20 mM SgcC3, and incubate at 37 �Cfor 1 h.

3. Terminate the reaction by adding 35 mL of 70% trichloroacetic acid

(TCA), and incubate on ice for 15 min to precipitate all proteins.

336 Shuangjun Lin et al.

4. Pellet the proteins by centrifugation at 4 �C and 14,000 rpm for 15 min,

wash the protein pellet twice with 200 mL of 5% cold TCA and once

with 200 mL of ice-cold ethanol, and dry the pellet in a speed-vac for

10 min.

5. Redissolve the protein pellet in 150 mL of 0.1 N KOH solution, and in-

cubate at 70 �C for 15 min to hydrolyze the SgcC2-tethered substrate

(S)-b-tyrosine and product (S)-3-chloro-b-tyrosine (Fig. 16.4B).6. Adjust the solution with 2 NHCl to�pH 6, cool on ice for 10 min, and

remove the precipitated proteins by centrifugation at 4 �C and

14,000 rpm for 15 min. Collect the supernatant, concentrate to dryness

in a speed-vac, and redissolve the residue in 50 mL of H2O.

7. Subject 20 mL of the sample from step 6 to HPLC analysis on an Apollo

C18 column (5 mM, 250�4.6 mm, Alltech Associates Inc., Deerfield,

IL) with UV detection at 280 nm. Elute the column at a flow rate of

1 mL/min with a 24-min linear gradient from 0% to 40% acetonitrile

in 0.1% TFA.

8. Determine the peaks corresponding to (S)-b-tyrosine and (S)-3-chloro-b-tyrosine by comparison to authentic standards (see Fig. 16.5B for a

representative HPLC chromatogram) and confirm their identity by

ESI-MS analysis.

2.2. In vitro characterization of SgcC-catalyzed hydroxylationof (S)-b-3-chloro-tyrosinyl-SgcC2

SgcC is a FAD-dependent monooxygenase, acting only on SgcC2-tethered

substrates, and requiring O2 and reduced FAD. The latter can be generated

by the C-1027 pathway-specific flavin reductase SgcE6 or E. coli flavin

reductase Fre from FAD and NADH.While (S)-3-chloro-b-tyrosyl-SgcC2is the natural substrate for SgcC in C-1027 biosynthesis (Fig. 16.3), both

(S)-3-bromo-b-tyrosyl-SgcC2 and (S)-3-iodo-b-tyrosyl-SgcC2 are better

substrates, with (S)-3-fluoro-b-tyrosyl-SgcC2, (S)-3-methyl-b-tyrosyl-SgcC2, and (S)-b-tyrosyl-SgcC2 also serving as substrates albeit significantlypoorer ones (Fig. 16.4C; Lin et al., 2008).

2.2.1 Expression in E. coli and overproduction and purification of SgcC1. Follow steps 1–8, Section 2.1.3, to clone sgcC from pBS1005 (Liu et al.,

2002), construct expression plasmid pBS1092, overproduce SgcC in

E. coli BL21 (DE3), and purify SgcC as an N-terminal His6-tagged fusion

protein (Lin et al., 2008).

337Tailoring Enzymes Acting on Carry Protein-Tethered Substrates

2. Check the purity of the isolated SgcC protein by SDS-PAGE on a

12% gel (Fig. 16.5A), determine the concentration by Bradford assay

(Bio-Rad), and store in 40% glycerol at �25 �C until use.

2.2.2 In vitro assay of SgcC-catalyzed hydroxylation of(S)-3-chloro-b-tyrosyl-SgcC2

1. Prepare (S)-3-chloro-b-tyrosyl-SgcC2 from (S)-3-chloro-b-tyrosineand holo-SgcC2 by taking advantage of the substrate promiscuity of

SgcC1 (Van Lanen et al., 2006; Fig. 16.4C). Steps 2–4, Section 2.1.2,

provide a protocol to prepare (S)-b-tyrosyl-SgcC2 from purified

holo-SgcC2 using SgcC1. An alternative protocol is provided in this sec-

tion for the preparation of (S)-3-chloro-b-tyrosyl-SgcC2 from apo-

SgcC2 directly by coupled assay using both Svp and SgcC1. The two

protocols afford comparative yields with>90% of the free (S)-b-tyrosineor analogues tethered to SgcC2 (Fig. 16.4).

2. Set up the in vitro 40-phosphopantetheinylation of apo-SgcC2 in 1.8 mL

of reaction solution containing 200 mM apo-SgcC2, 1.0 mM CoA,

12.5 mM MgCl2, and 2.0 mM TCEP in 100 mM Tris–HCl (pH 7.5),

at 25 �C. Initiate the reaction by adding 10 mM Svp, and incubate at

25 �C for 45 min.

3. Prepare a loading solution containing 7.0 mM (S)-3-chloro-b-tyrosine,8 mM ATP, 2.0 mM TCEP, and 12.5 mMMgCl2 in 100 mM Tris–HCl

(pH 7.5), and mix it with an equal volume of the holo-SgcC2 reaction

solution from step 2. Initiate the loading reaction by adding 5 mM SgcC1,

and incubate at 25 �C for 1 h. Follow steps 3 and 4, Section 2.1.2, to

purify (S)-3-chloro-b-tyrosyl-SgcC2.4. Set up the SgcC-catalyzed hydroxylation of (S)-3-chloro-b-tyrosyl-

SgcC2 in 200 mL of reaction solution containing 250 mM (S)-3-

chloro-b-tyrosyl-SgcC2, 5 mM NADH, 10 mM FAD, 1 mM TCEP,

50 mM NaCl, and 5 mM SgcC in 50 mM sodium phosphate (pH 6.0),

at 25 �C.5. Initiate the reactionsby adding1.5 mMSgcE6and incubate at 25 �Cfor1 h.

6. Terminate the reaction and recover (S)-3-chloro-b-tyrosyl-SgcC2 and

its hydroxylated product (S)-3-chloro-5-hydroxy-b-tyrosyl-SgcC2 by

following the steps 3 and 4, Section 2.1.5.

7. Redissolve the protein pellet from step 6 by adding first 5 mL of 1.5 M

DTT and then 150 mL of 0.1N KOH, and incubate at 50 �C for

15 min to hydrolyze the SgcC2-tethered substrate (S)-3-chloro-b-tyrosine and product (S)-3-chloro-5-hydroxy-b-tyrosine (Fig. 16.4C).

338 Shuangjun Lin et al.

8. Follow steps 6–8, Section 2.1.5, for sample preparation and HPLC anal-

ysis. Determine the peaks corresponding to (S)-3-chloro-b-tyrosine and(S)-3-chloro-5-hydroxy-b-tyrosine by comparison to authentic stan-

dards (see Fig. 16.5C for a representative HPLC chromatogram), and

confirm their identity by ESI-MS analysis.

2.3. Exploitation of SgcC2-tethered (S)-b-tyrosine analoguesfor structural diversification

2.3.1 SgcC3-catalyzed bromination of (S)-b-tyrosyl-SgcC21. Prepare SgcC3 according to Section 2.1.3 and SgcE6 according to Sec-

tion 2.1.4 with the exception of excluding NaCl in all buffers used for

their purification.

2. Prepare the (S)-b-tyrosyl-SgcC2 according to Section 2.1.2.

3. Desalt the (S)-b-tyrosyl-SgcC2 sample from step 2 using a Superose 12

column (GE Healthcare) in 20 mM sodium phosphate (pH 7.0), and run

the sample twice to ensure the complete removal of residual NaCl.

4. Set up the SgcC3-catalyzed bromination of (S)-b-tyrosyl-SgcC2 reac-

tion in an identical condition to that of chlorination with the exception

of replacing NaCl with 0.1 MNaBr and excluding TCEP from the assay

solution, and follow the steps in Section 2.1.5 to carry out the reaction

and analyze the product (Fig. 16.4B). Determine the formation of (S)-3-

bromo-b-tyrosine by HPLC analysis and comparison with authentic

standard (see Fig. 16.5D for a representative HPLC chromatogram),

and confirm its identity by ESI-MS analysis.

2.3.2 SgcC-catalyzed hydroxylation of SgcC2-tethered (S)-b-tyrosineanalogues

1. Prepare SgcE6 according to Section 2.1.4 and SgcC according to

Section 2.2.2.

2. Prepare SgcC2-tethered b-tyrosine analogues of (S)-3-fluoro-b-tyrosyl-SgcC2, (S)-3-bromo-b-tyrosyl-SgcC2, (S)-3-iodo-b-tyrosyl-SgcC2,and (S)-3-methyl-b-tyrosyl-SgcC2 according to Section 2.1.2 with

the exception of replacing (S)-b-tyrosine with corresponding analogues

(Fig. 16.4C).

3. Since SgcC hydroxylates SgcC2-tethered (S)-b-tyrosine analogues withvarying rates, the assay condition described for (S)-3-chloro-b-tyrosyl-SgcC2 in Section 2.2.2 needs optimization for each of the analogues

to ensure efficient formation of the hydroxylated products.

339Tailoring Enzymes Acting on Carry Protein-Tethered Substrates

4. Set up the SgcC-catalyzed hydroxylation reaction in 200 mL of solution

containing 250 mM SgcC2-tethered (S)-b-tyrosine or analogues, 5 mM

NADH, 10 mM FAD, 1 mM TCEP, and 50 mM NaCl, in 50 mM so-

dium phosphate (pH 6.0) at 25 �C. For (S)-3-bromo-b-tyrosyl-SgcC2and (S)-3-iodo-b-tyrosyl-SgcC2, add 1.5 mM SgcC and 2 mM SgcE6

and incubate the reaction at 25 �C for 20 min, while for (S)-3-

methyl-b-tyrosyl-SgcC2, (S)-3-fluoro-b-tyrosyl-SgcC2, and (S)-b-tyrosyl-SgcC2, add 6 mM SgcC and 2 mM SgcE6 and incubate the

reaction at 25 �C for 1 h.

5. Terminate the reaction, recover SgcC2-tethered substrates and their

hydroxylated products, release them from SgcC2 by hydrolysis, and

determine their identities by HPLC and ESI-MS analyses by following

the steps 6–8, Section 2.2.2 (Fig. 16.4C). For maximal sensitivity, use

varying wavelengths to detect the formation of each of the hydroxylated

products: (S)-3-fluoro-b-5-hydroxy-tyrosine from (S)-3-fluoro-b-tyrosyl-SgcC2 at UV 272 nm, (S)-3-bromo-5-hydroxy-b-tyrosine from(S)-3-bromo-b-tyrosyl-SgcC2 at UV 282 nm, (S)-3-iodo-5-hydroxy-

b-tyrosine from (S)-3-iodo-b-tyrosyl-SgcC2 at UV 284 nm, (S)-3-

methyl-5-hydroxy-b-tyrosine from (S)-3-methyl-b-tyrosyl-SgcC2 at

UV 278 nm, and (S)-3-hydroxy-b-tyrosine from (S)-b-tyrosyl-SgcC2at UV 277 nm.

3. CONCLUSION

We highlighted in this chapter the emerging roles CPs play in precursor

biosynthesis and post-PKS or post-NRPS modifications and summarized tai-

loring enzymes that are known to act on CP-tethered substrates (Figs. 16.1

and 16.2; Table 16.1). By covalently tethering, CPs sequester the substrates

from endogenous metabolite pools, thereby increasing their concentration

at the active sites for catalysis. CPs also provide the critical protein–protein

recognitions among the various enzymatic partners, and this feature

provides a new opportunity to engineer natural product diversity by

exploiting CPs to increase substrate promiscuity for the tailoring steps.

Realization of the full potential of tailoring enzymes that act on CP-

tethered substrates in engineered biosynthesis of natural product structural

diversity depends on continued discovery of new members of this family

of enzymes, further expansion of the catalytic portfolio, fundamental char-

acterization of their reaction mechanisms, and exploitation of their

340 Shuangjun Lin et al.

portability in the broad context of natural product biosynthetic machinery.

The protocols provided here were developed from our current effort to

characterize the SgcC3 halogenase and SgcC hydroxylase, acting exclusively

on SgcC2-tethered b-tyrosine and analogues, in the biosynthesis of the (S)-

3-chloro-5-hydroxy-b-tyrosine moiety of the antitumor antibiotic C-1027

(Van Lanen & Shen, 2008), but should be applicable to mechanistic charac-

terization and engineered exploitation of other tailoring enzymes that act on

CP-tethered substrates in natural product biosynthesis and structural diver-

sification. The ultimate goal would be to use the in vitro findings to guide

in vivo engineering to produce designer natural product analogues. For

example, it has already been demonstrated that variants of the b-tyrosinemoiety can be tolerated by the C-1027 biosynthetic machinery, resulting

in the production of several C-1027 analogues (Fig. 16.3; Kennedy et al.,

2007; Van Lanen et al., 2005). It would be fascinating to investigate if

the sets of b-tyrosine analogues that can be readily generated by SgcC3

and SgcC in vitro (Fig. 16.4) can be recapitulated in vivo to produce a

focused library of C-1027 analogues, some of which could be developed

into novel anticancer drugs.

ACKNOWLEDGMENTThis work was supported in part by National Institute of Health (NIH) grant CA078747.

REFERENCESBlasiak, L. C., Vaillancourt, F. H., Walsh, C. T., & Drennan, C. L. (2006). Crystal structure

of the non-haem iron halogenase in syringomycin biosynthesis. Nature, 440, 368–371.Chan, D. I., & Vogel, H. J. (2010). Current understanding of fatty acid biosynthesis and the

acyl carrier protein. The Biochemical Journal, 430, 1–19.Chen, H., Hubbard, B. K., O’Connor, S. E., & Walsh, C. T. (2002). Formation of

b-hydroxy histidine in the biosynthesis of nikkomycin antibiotics. Chemistry & Biology,9, 103–112.

Chen, H., & Walsh, C. T. (2001). Coumarin formation in novobiocin biosynthesis:b-Hydroxylation of the aminoacyl enzyme tyrosyl-S-NovH by a cytochrome P450NovI. Chemistry & Biology, 8, 301–312.

Cheng, Y.-Q., Coughlin, J. M., Lim, S.-K., & Shen, B. (2009). Type I polyketide synthasesthat require discrete acyltransferases. Methods in Enzymology, 459, 165–186.

Christenson, S. D., Liu, W., Toney, M. D., & Shen, B. (2003). A novel4-methylideneimidazole-5-one-containing tyrosine aminomutase in enediyne antitumorantibiotic C-1027 biosynthesis. Journal of the American Chemical Society, 125, 6062–6063.

Christenson, S. D.,Wu,W., Spies, M. A., Shen, B., & Toney, M. D. (2003). Kinetic analysisof the 4-methylideneimidazole-5-one-containing tyrosine aminomutase in enediyneantitumor antibiotic C-1027 biosynthesis. Biochemistry, 42, 12708–12728.

Cryle, M. J., Meinhart, A., & Schlichting, I. (2010). Structural characterization of OxyD,a cytochrome P450 involved in b-hydroxytyrosine formation in vancomycin biosynthe-sis. The Journal of Biological Chemistry, 285, 24562–24574.

341Tailoring Enzymes Acting on Carry Protein-Tethered Substrates

Dorrestein, P. C., Yeh, E., Garneau-Tsodikova, S., Kelleher, N. L., & Walsh, C. T. (2005).Dichlorination of a pyrrolyl-S-carrier protein by FADH2-dependent halogenase PltAduring pyoluterin biosynthesis. Proceedings of the National Academy of Sciences of the UnitedStates of America, 102, 13843–13848.

Du, L., Sanchez, C., & Shen, B. (2001). Biosynthesis of hybrid peptide and polyketidemetabolites: Prospects towards engineering novel molecules. Metabolic Engineering, 3,78–95.

Fischbach, M. A., & Walsh, C. T. (2006). Assembly-line enzymology for polyketide andnonribosomal peptide antibiotics: Logic, machinery, and mechanisms.Chemistry Review,106, 3468–3496.

Fischbach, M. A., &Walsh, C. T. (2010). Natural products version 2.0: Connecting genes tomolecules. Journal of the American Chemical Society, 132, 2469–2493.

Flatt, P. M., O’Connell, S. J., McPhail, K. L., Zeller, G., Willis, C. L., Sherman, D. H., et al.(2006). Characterization of the initial enzymatic steps of barbamide biosynthesis. Journalof Natural Products, 69, 938–944.

Gago, G., Diacovich, L., Arabolaza, A., Tsai, S.-C., & Gramajo, H. (2011). Fatty acid bio-synthesis in actinomycetes. FEMS Microbiology Reviews, 35, 475–497.

Galonic, D. P., Vaillancourt, F. H., &Walsh, C. T. (2006). Halogenation of unactivated car-bon centers in natural product biosynthesis: Trichlorination of leucine during barbamidebiosynthesis. Journal of the American Chemical Society, 128, 3900–3901.

Garneau-Tsodikova, S., Dorrestein, P. C., Kelleher, N. L., & Walsh, C. T. (2005). Charac-terization of the formation of the pyrrole moiety during clorobiocin and coumermycinA1 biosynthesis. Biochemistry, 44, 2770–2780.

Giessen, T. W., Kraas, F. I., & Marahiel, M. A. (2011). A four-enzyme pathway for3,5-dihydroxy-4-methylanthranilic acid formation and incorporation into the antitumorantibiotic sibiromycin. Biochemistry, 50, 5680–5692.

Hollenhorst, M. A., Bumpus, S. B., Matthews, M. L., Jr., Bollinger, J. M., Kelleher, N. L., &Walsh, C. T. (2010). The nonribosomal peptide synthetase enzyme DdaD tethers Nb-fumaramoyl-L-2,3-diaminopropionate for Fe(II)/a-ketoglutarate-dependent epoxida-tion by DdaC during dapdiamide antibiotic biosynthesis. Journal of the American ChemicalSociety, 132, 15773–15781.

Horsman, G. P., Van Lanen, S. G., & Shen, B. (2009). Iterative type I polyketide synthases forenediyne core biosynthesis. Methods in Enzymology, 459, 97–112.

Jiang, W., Heemstra, J. R., Jr., Forseth, R. R., Neumann, C. S., Manaviazar, S.,Schroeder, F. C., et al. (2011). Biosynthetic chlorination of the piperazate residue inkutzneride biosynthesis by KthP. Biochemistry, 50, 6063–6072.

Jiang, H., Rajski, S. R., & Shen, B. (2009). Tandem acyl carrier protein domains in poly-unsaturated fatty acid synthases. Methods in Enzymology, 459, 79–96.

Kennedy, D. R., Gawron, L. S., Ju, J., Liu, W., Shen, B., & Beerman, T. A. (2007). Singlechemical modifications of the C-1027 enediyne core, a radiomimetic antitumor drug,affect both drug potency and the role of ataxia-telangiectasia mutated in cellular responsesto DNA double-strand breaks. Cancer Research, 67, 773–781.

Kopp, F., Linne, U., Oberthur, M., & Marahiel, M. A. (2008). Harnessing the chemicalactivation inherent to carrier protein-bound thioesters for the characterization oflipopeptide fatty acid tailoring enzymes. Journal of the American Chemical Society, 130,2656–2666.

Lambalot, R. H., Gehring, A. M., Flugel, R. S., Zuber, P., LaCelle, M., Marahiel, M. A.,et al. (1996). A new enzyme superfamily—The phosphopantetheinyl transferase. Chem-istry & Biology, 3, 923–936.

Lin, S., Huang, T., Horsman, G. P., Huang, S.-X., Guo, X., & Shen, B. (2012). Specificity ofthe ester bond forming condensation enzyme SgcC5 in C-1027 biosynthesis.Organic Let-ters, 14, 2300–2303.

342 Shuangjun Lin et al.

Lin, S., Van Lanen, S. G., & Shen, B. (2007). Regiospecific chlorination of (S)-beta-tyrosyl-S-carrier protein catalyzed by SgcC3 in the biosynthesis of the enediyne antitumor an-tibiotic C-1027. Journal of the American Chemical Society, 129, 12432–12438.

Lin, S., Van Lanen, S. G., & Shen, B. (2008). Characterization of the two-component, FAD-dependent monooxygenase SgcC that requires carrier protein-tethered substrates for thebiosynthesis of the enediyne antitumor antibiotic C-1027. Journal of the American ChemicalSociety, 130, 6616–6623.

Lin, S., Van Lanen, S. G., & Shen, B. (2009). A free-standing condensation enzyme catalyz-ing ester bond formation in C-1027 biosynthesis. Proceedings of the National Academy ofSciences of the United States of America, 106, 4183–4188.

Liu, W., Christenson, S. D., Standage, S., & Shen, B. (2002). Biosynthesis of the enediyneantitumor antibiotic C-1027. Science, 297, 1170–1173.

Makris, T. M., Chakrabarti, M., Munck, E., & Lipscomb, J. D. (2010). A family of diironmonooxygenase catalyzing amino acid beta-hydroxylation in antibiotic biosynthersis.Proceedings of the National Academy of Sciences of the United States of America, 107,15391–15396.

Marahiel, M. A., & Essen, L.-O. (2009). Nonribosomal peptide synthetases: Mechanistic andstructural aspects of essential domains. Methods in Enzymology, 458, 337–351.

Mercer, A. C., & Burkart, M. D. (2007). The ubiquitous carrier protein—A window to me-tabolite biosynthesis. Natural Product Reports, 24, 750–773.

Mo, S. J., Kim, D. H., Lee, J. H., Park, J. W., Basnet, D. B., Ban, Y. H., et al. (2011).Biosynthesis of the allylmalonyl-CoA extender unit for the FK506 polyketide synthaseproceeds through a dedicated polyketide synthase and facilitates the mutasynthesis ofanalogues. Journal of the American Chemical Society, 133, 976–985.

Neumann, C. S., & Walsh, C. T. (2008). Biosynthesis of (�)-(1S,2R)-allocoronamic acylthioester by an FeII-dependent halogenase and a cyclopropane-forming flavoprotein.Journal of the American Chemical Society, 130, 14022–14023.

Pacholec, M., Hillson, N. J., &Walsh, C. T. (2005). NovJ/NovK catalyze benzylic oxidationof a b-hydroxyl tyrosyl-S-pantetheinyl enzyme during aminocoumarin ring formation innovobiocin biosynthesis. Biochemistry, 44, 2819–2826.

Reimmann, C., Patel, H. M., Serino, L., Barone, M., Walsh, C. T., & Haas, D. (2001).Essential PchG-dependent reduction in pyochelin biosynthesis of Pseudomonas aeruginosa.Journal of Bacteriology, 183, 813–820.

Sanchez, C., Du, L., Edwards, D. J., Toney, M. D., & Shen, B. (2001). Cloning and char-acterization of a phosphopantetheinyl transferase from Streptomyces verticillusATCC15003, the producer of the hybrid peptide-polyketide antitumor drug bleomycin.Chemistry & Biology, 8, 725–738.

Shen, B. (2000). Aromatic polyketide biosynthesis. Topics in Current Chemistry, 209, 1–51.Shen, B. (2003). Polyketide biosynthesis beyond the type I, II, and III polyketide synthase

paradigms. Current Opinion in Chemical Biology, 7, 285–295.Singh, G. M., Fortin, P., Koglin, A., & Walsh, C. T. (2008). Beta hydroxylation of the

aspartyl residue in the phytotoxin syringomycin E: Characterization of two candidatehydroxylases AspH and SyrP in Pseudomonas syringae. Biochemistry, 47, 11310–11320.

Staunton, J., & Weissman, K. J. (2001). Polyketide biosynthesis: A millennium review.Natural Product Reports, 18, 380–416.

Strieker, M., Nolan, E. M., Walsh, C. T., &Marahiel, M. A. (2009). Stereospecific synthesisof threo- and erythro-b-hydroxyglutamic acid during kutzneride biosynthesis. Journal of theAmerican Chemical Society, 131, 13523–13530.

Thomas, M. G., Burkart, M. D., &Walsh, C. T. (2002). Conversion of L-proline to pyrrolyl-2-carboxyl-S-PCP during undecylprodigiosin and pyoluterin biosynthesis. Chemistry &Biology, 9, 171–184.

343Tailoring Enzymes Acting on Carry Protein-Tethered Substrates

Ueki, M., Galonic, D. P., Vaillancourt, F. H., Garneau-Tsodikova, S., Yhe, E.,Vosburg, D. A., et al. (2006). Enzymatic generation of the antimetabolite g,g-dichloroaminobutyrate by NRPS and mononuclear iron halogenase action in a strep-tomycete. Chemistry & Biology, 13, 1183–1191.

Vaillancourt, F. H., Yeh, E., Vosburg, D. A., O’Connor, S. E., &Walsh, C. T. (2005). Cryp-tic chlorination by a non-haem iron enzyme during cyclopropyl amino acid biosynthesis.Nature, 436, 1191–1194.

Vaillancourt, F. H., Yin, J., &Walsh, C. T. (2005). SyrB2 in syringomycin E biosynthesis is anonheme FeII a-ketoglutarate- and O2-dependent halogense. Proceedings of the NationalAcademy of Sciences of the United States of America, 102, 10111–10116.

Van Lanen, S. G., Dorrestein, P. C., Christenson, S. D., Liu, W., Ju, J., Kelleher, N. L., et al.(2005). Biosynthesis of the b-amino acidmoiety of enediyne antitunor antibiotic C-1027featuring b-amino acyl-S-carrier protein intermediates. Journal of the American ChemicalSociety, 127, 11594–11595.

Van Lanen, S. G., Lin, S., Dorrestein, P. C., Kelleher, N. L., & Shen, B. (2006). Substratespecificity of the adenylation enzyme SgcC1 involved in the biosynthesis of the enediyneantitumor antibiotic C-1027. The Journal of Biological Chemistry, 281, 29633–29640.

Van Lanen, S. G., & Shen, B. (2008). Biosynthesis of enediyne antitumor antibiotics. CurrentTopics in Medicinal Chemistry, 8, 448–459.

Walsh, C. T., Chen, H., Keating, T. A., Hubbard, B. K., Losey, H. C., Luo, L., et al. (2001).Tailoring enzymes that modify nonribosomal peptides during and after chain elongationon NRPS assembly lines. Current Opinion in Chemical Biology, 5, 525–534.

Weissman, K. J. (2009). Introduction to polyketide biosynthesis.Methods in Enzymology, 459,3–16.

Zhang, W., Ntai, I., Bolla, M., Malcolmson, S. J., Kahne, D., Kelleher, N. L., et al. (2011).Nine enzymes are required for assembly of the pacidamycin group of peptidyl nucleosideantibiotics. Journal of the American Chemical Society, 133, 5240–5243.