Chapter 1

Review of Literature

Chapter 1

Fatty acid synthases (F ASs) and polyketide synthases (PKSs) constitute

two enzymatic systems that possess many similarities including a common reaction

mechanism and similar substrate utilization. Yet, they are programmed to perform

very different functions in an organism. While the F AS enzymes are involved in

primary metabolism for the production of saturated fatty acids, PKSs have been

typically characterized from Streptomyces and are responsible for the biosynthesis

of a wide range of complex natural products (Hopwood, 1997; Katz and Donadio,

1993; O'Hagan, 1992; Sanchez et aI., 2008; Schweizer and Hofmann, 2004).

Classically, the biosynthetic machineries for fatty acids and polyketides have been

studied independently and the channeling of fatty acids into the PKS enzymatic

machinery was believed to occur through a CoA-dependent activation mechanism.

Recent characterization of fatty acyl-AMP ligases (FAALs) has provided an

alternate link between these two classes of enzymes (Trivedi et ai., 2004; Hansen

et ai., 2007). F AALs catalyze the activation of fatty acids as fatty acy l-adeny lates,

which are subsequently transferred as starter substrates onto PKS enzymes. These

enzymes have now been characterized from a number of organisms (Arora et ai.,

2009; Hansen et ai., 2007).

Sequencing of the H37Rv strain of Mycobacterium tuberculosis (Mtb)

revealed a number of enzymes involved in lipid metabolism (Cole et aI., 1998). A

striking feature of the sequencing results was the identification of genes

homologous to PKSs. Cell free reconstitution of some of these PKSs, along with

gene inactivation studies in mycobacteria have shown the involvement of these

genes in the biosynthesis of cell wall lipids (Gokhale et aI., 2007b; Jackson et aI.,

2007). These lipids are present as a complex network of sugars and proteins in the

- 4-

Chapter 1

cell wall and have also been implicated in the pathogenicity of Mtb (Brennan and

Nikaido, 1995).

This chapter discusses the F AS and PKS enzymatic systems and highlights

the similarities and differences between these two megasynthase families. The

recent advances made towards understanding the three dimensional organization of

these enzymes are also described. The chapter ends with a discussion on the

chemical composition of the mycobacterial cell envelope and the current

knowledge of the role played by PKSs in the biosynthesis of various cell envelope

lipids.

1.1 FATTY ACID SYNTHASES

Fatty acids are compounds that contain a long hydrocarbon chain ending in

a carboxylate group. These acids play an important role in the physiology of an

organism and apart from acting as an energy source act as building blocks of

several metabolic compounds. Studies focused on finding the origin of these fatty

acids date back to 1878, when Nencki proposed that acetaldehyde coming from

degradation of glucose derived lactate acts as a precursor of fatty ac,ids (Nencki,

1878). It was suggested that two acetaldehyde units follow an aldol condensation

reaction to yield a ~-hydroxy aldehyde, which undergoes further rearrangements to

yield butyric acid. Stanley Raper in 1907 also reported that fatty acids are

produced by the condensation of some highly reactive substance containing two

carbons (Raper, 1907). His studies were unable to shed light on the nature of the

reactive substance but he predicted that the precursor unit could either be ethanol

or acetaldehyde or acetic acid. These hypotheses could not be tested until the

middle of the nineteenth century, due to lack of suitable techniques to investigate

- 5 -

Chapter 1

into fatty acid biosynthesis. In 1944, Rittenberg and Bloch used isotopically

labelled acetate (which was labelled in the carboxyl-group with 13C and in the

methyl group with deuterium) to study fatty acid biosynthesis (Rittenberg, 1944).

Labelled acetate was fed to rats and the body fat was analyzed for accumulation of

both the isotopes. Their studies demonstrated that fatty acids were indeed

synthesized by acetate derived C2 units. However, it was always suspected that the

actual metabolic intermediate was not acetate but some activated form of acetate.

Discovery of coenzyme A (CoA) in Fritz Lipmann's lab in early 1950's and the

experiments that followed ultimately led to the conclusion that the activated form

of acetate was in fact acetyl-CoA (Kaplas and Lipmann, 1948; Klein and Lipmann,

1953a; Klein and Lipmann, 1953b).

While the hunt for finding the origin of fatty acids was on, a number of

research groups focused on studying the degradation of fatty acids in various

organisms. Experiments involving usage of 'chemical tracers' enabled Knoop to

conclude that fatty acid catabolism requires oxidation at the ~-carbon atom with a

loss of C2 unit (Fruton, 1972). This idea was supported by an experiment by Bloch

and Rittenberg in 1944, when they found that isotope-labelled fatty acids gave a

product that could carry out acetylation reactions similar to acetic acid (Bloch and

Rittenberg, 1944). They concluded that the ~-oxidation product was either acetic

acid or a functional derivative thereof. These results were supported by a number

of other experiments and led to the notion that fatty acid biosynthesis proceeded by

reversal of the mitochondrial pathway for ~-oxidation of fatty acids. However, this

theory of biosynthesis being reverse of degradation was put to test when several

research groups demonstrated biosynthesis of fatty acid by mitochondria-free

- 6-

Chapter 1

cytosolic extracts (Brady and Gurin, 1952; Popjak and Tietz, 1955; Tietz and

Popjak, 1955). Further insights into fatty acid biosynthesis were provided in 1958

~rady and Wakil independently published that acetyl-CoA is first converted \, ---

to malonyl-CoA which undergoes a decarboxylative condensation reaction to form

fatty acids (Brady, 1958; Wakil, 1958). This gave rise to two independent routes

of fatty acid biosynthesis: non mitochondrial or malonyl CoA pathway and the

mitochondrial elongation system. The malonyl CoA pathway was found to be the

major route of fatty acid biosynthesis in animals and much of the research on fatty

acid biosynthesis focused on this cytosolic pathway (Wakil, 1961). In fact, details

of the mitochondrial pathway have only been recently elucidated and the enzymes

involved in this pathway are characterized to be free standing, mono-functional

proteins analogous to what is now called the type II F AS systiem (Miinalainen et

aI., 2003; Zhang et aI., 2005; Zhang et aI., 2003).

A landmark discovery in the cytosolic pathway of fatty acid biosynthesis

was the discovery of a small heat-stable protein called acyl carrier protein (ACP).

It was found that a serine residue in the protein was post-translationally modified

with a phosphopantetheinyl moiety that functioned as a carrier arm for various

reaction intermediates (Majerus et aI., 1964; Sauer et aI., 1964). By late 1960s,

F AS proteins were purified from a number of organisms and were either found to

contain all catalytic sites on a single high molecular mass protein or were found as

a dissociated system wherein each catalytic site was present on a separate

polypeptide. The two F AS systems were identified as F AS I (high molecular mass

FASs) and FAS II (free standing proteins) systems of fatty acid synthesis (Brindley

et aI., 1969). It is now known that the prototypical F AS I is found in mammals and

- 7 -

Chapter 1

consists of a single gene that produces a polypeptide, which contains all of the

reaction centers required to biosynthesize fatty acids. In lower eukaryotes, such as

yeast, there are two genes, and their polypeptide products coalesce to form a

multifunctional complex. F AS II system is found in bacteria, plants, and parasites.

Interestingly, while the F AS I system usually produces only palmitic acid, the F AS

II system is capable of producing a wide range of products for cellular metabolism

(Schweizer and Hofmann, 2004).

1.1.1 Type II fatty acid synthases

The F AS II pathway has been majorly established in Escherichia coli,

which serves as a model system to understand the type II F AS systems in other

organisms (Cronan, 1996). The basic steps in the fatty acid synthesis cycle are

common to all bacteria, and the genes encoding the enzymes are highly conserved.

However, there are cases, where differences from the E. coli model can be

observed (Marrakchi et ai., 2002b; White et ai., 2005). Biosynthesis of fatty acids

by the Type II FAS system involves·non covalent interactions between a series of

proteins that carry individual catalytic sites and are each encoded by a discrete

gene. The intermediate acyl chains are shuttled between various catalytic proteins

as thioesters of ACP. The F AS II pathway is outlined in figure 1.1.

The first enzyme of the FAS II system is acetyl-CoA carboxylase (ACC),

which catalyzes conversion of acetyl-CoA molecules to malonyl-CoA. The

malonyl-CoA thus produced, is transferred to the ACP by a malonyl-CoA: ACP

transcylase (FabD) leading to the formation of malonyl-ACP (Figure 1.1). This

then undergoes a condensation reaction with acetyl-CoA to form ~-ketoacyl-ACP

and CO2• The condensation reaction is catalyzed by ~-ketoacyl-ACP synthase HI

- 8 -

Chapter 1

(FabH). The acetoacetyl-ACP fonned by FabH then enters the elongation cycle,

where the acyl chain attached to the ACP is extended by two carbons with each

successive round of condensation.

o H,CP-SCoA

Acetyl-CoA (31 o 0

·O~SCoA MaIonyloCoA

B1G o

·o~( Acp~1 ',-I ~ Malonyl·ACP

COz

~~/~B :.~= ~.- ;6'~1

~(~I f.:\ ~:; ~-Hydr()xyacyl-ACP ~ AcyI-ACP

( F~ [jJabl NAD(P)· FaI>Z 0 r. FabK

",c~c:::J FabL

Enoy~ACP NAO(P)H • H"

Figure 1.1: The FAS II biosynthetic pathway.

Four core enzyme activities are responsible for progressive chain extension

and reduction of the B-keto group to complete saturation. The NADPH-dependent

reduction of the B-keto group to fonn B-hydroxyacyl-ACP is catalyzed by B-

ketoacyl-ACP reductase (FabG) and is followed by a dehydration step catalyzed by

B-hydroxyacyl-ACP dehydratases (either FabA or FabZ). The last NADPH-

dependent reductive step of conversion of enoyl-ACP to acyl-ACP is mediated by

enoyl-ACP reductase I, II or III (FabI, FabK or FabL, respectively). Subsequent

rounds of elongation and are catalyzed by the condensing enzymes FabB or FabF.

- 9 -

Chapter 1 ---------------------------------------------------------

1.1.1.1 Acyl-CoA carboxylases (ACCs)

ACCs catalyze the first step in F AS II biosynthetic machinery and play a

key role in the regulation of fatty acid biosynthesis. ACCs are multi-subunit

proteins in prokaryotic organisms, whereas in most eukaryotes, they exist as large,

multi-domain enzymes (Cronan and Waldrop, 2002). These enzymes catalyze the

biotin-dependent carboxylation of acetyl-CoA to malonyl-CoA via two partial

reactions (Figure 1.2).

ATP+~+Pi

Biotin carboxylase

O~ 0 ... S~BCCP ... s~B<XP H" ACCs H"·

rNlfN-l _o,pNlfN-l o "-- - 0

Biotin-BCCP Crbo ~ I t f Carboxybiotin-BCCP a xy rans erase

~o o H:JcJlQ)A

.o"c.}J... Acetyl-COA Malonyl-GoA

Figure 1.2: The ACC catalyzed biotin carboxylation transferase reaction.

The first reaction involves the phosphorylation of bicarbonate by ATP to

form a carboxy-phosphate intermediate. This is followed by transfer of the

carboxyl group to the biotin of the biotin carrier protein (BCCP) to generate the

carboxybiotin. The biotin is attached to the protein via an amide bond between the

valeric acid side chain of biotin and the £-amino group of a specific lysine residue.

In the second reaction, the carboxyl group is transferred from biotin to the acetyl-

CoA substrate forming malonyl-CoA product. In Actinomycetes, these activities

are distributed in two polypeptide chains: u- (for biotin carboxylation) and ~- (for

carboxyl transfer) (Ertle, 1973; Henrikson and Allen, 1979; Hunaiti and

- 10 -

Chapter 1

Kolattukudy, 1982). Recent studies have identified an additional E-subunit

required for complex formation between the u- and the p-subunits and maintaining

optimal activity of the resulting ACCase in Streptomyces (Diacovich et aI., 2002;

Rodriguez et aI., 2001; Rodriguez and Gramajo, 1999). In addition to its role as a

substrate in fatty acid biosynthesis, malonyl-CoA also plays a regulatory role in

controlling mitochondrial fatty acid uptake through allosteric inhibition of camitine

palmitoyltransferase (CPT-I), the enzyme catalyzing the first committed step in

mitochondrial fatty acid oxidation (Rasmussen et aI., 2002).

1.1.1.2 Acyl Carrier Protein (ACP)

ACPs play an important role in fatty acid biosynthesis by shuttling the

intermediate chains between various catalytic sites of the F AS. These proteins are

a group of highly related, small acidic proteins with molecular weights of about 10

kDa. Generally, they have a high helical content and readily fold into their native

conformation following heat-induced or pH-induced denaturation (Rock and

Cronan, 1979). ACPs are produced as apoproteins in the cell and are converted to

their active form by the action of an enzyme called ACP synthase (AcpS)

(Jackowski and Rock, 1984; Powell et aI., 1969). AcpS catalyzes the transfer of

the 4' -phosphopantetheine (p-pant) prosthetic group of CoA on to apo-ACP for the

formation of the catalytically active holo-ACP. A recent report suggested that

while AcpS is involved in the transfer of p-pant group onto F AS proteins, another

p-pant transferase called PptT is involved in activation of PKS and NRPS enzymes

in mycobacteria (Chalut et aI., 2006). Many homologues of these enzymes have

been identified in other organisms (Bobrov et aI., 2002; Garcia-Estrada et aI.,

2008; Neville et aI., 2005; Venkitasubramanian et aI., 2007; Zhang et at, 2003).

- 11 -

Chapter 1

The ACP involved in the type II F AS system of Mtb is called AcpM and

plays a key role in the biosynthesis of mycolic acids (Barry et ai., 1998; Kremer et

ai., 2001; Schaeffer et ai., 200la). This enzyme has a C-terminal extension which

exists as an unfolded domain. It is believed that this extension is either involved in

protein-protein interactions or in sequestration of the long acyl chains from solvent

during mycolic acid biosynthesis (Barry et ai., 1998; Wong et ai., 2002).

1.1.1.3 Malonyl-CoA:ACP Transacylase (FabD)

FabD is responsible for the transfer of the malonyl moiety from malonyl-

CoA to the terminal sulfhydryl group of ACP. This enzyme uses a ping-pong

kinetic mechanism to load the malonyl moiety onto the serine residue within the

GHSLG motif (Ruch and Vagelos, 1973). In the forward reaction, FabD first

binds malonyl-CoA and takes up the malonyl moiety with the release of Co A from

the enzyme. This is followed by the binding of ACP and transfer of the malonyl-

group to the p-pant arm of ACP. An important feature of the FabD proteins is the

stable acyl enzyme intermediate that is formed during the reaction. This is

attributed to the lack of an ideally positioned oxyanion hole in these proteins. The

oxyanion hole, if present, would stabilize the transition state for the hydrolytic

reaction and lead to the cleavage of the acyl enzyme intermediate via nucleophilic

attack by a water molecule (White et ai., 2005).

1.1.1.4 Condensing Enzymes: p-Kefoacyl-ACP Synthase I, II and III (FabB, FabF and FabH)

Condensing enzymes catalyze the chain-initiation and chain-elongation

steps of fatty acid synthesis. FabH catalyzes the initiation step in acyl chain

formation and uses acyl-CoA as a primer to catalyze a condensation reaction with

- 12 -

Chapter 1

malonyl-ACP (Jackowski et aI., 1989). FabB and FabF are components of the

elongation cycle and condense an acyl-ACP intermediate with malonyl-ACP. All

three enzymes belong to the thiolase superfamily of proteins and the core structure

consists of two ~a~a~a~~ motifs related by pseudo dyad duplication. While FabH

proteins have a Cys-Asn-His catalytic triad, the FabBIF proteins have a Cys-His-

His configuration in the active site (White et aI., 2005). It is believed that the

substrate specificities and expression level of FabB and FabF determines the

structure and distribution of fatty acid products in an organism (Marrakchi et aI.,

2002b).

During FabH catalysis, the starter acyl group is first transferred to the

active site sulfhydryl group leading to release of CoA. This is followed by the

binding of malonyl-ACP and condensation of the two acyl chains to release ~­

ketoacyl-ACP and C02. FabH substrate specificity is a major determining factor in

membrane fatty acid composition. In Bacillus subtilis, the BsFabH proteins prefer

branched chain acyl-CoA substrates derived from amino acid metabolism, leading

to the production of primarily iso- and anteisobranched fatty acids (Choi et aI.,

2000b; Han et aI., 1998; Lu et aI., 2004). Similarly, in Mtb, the MtFabH prefers

FAS I derived long chain acyl-CoA substrates (Choi et aI., 2000b). Analogous to

FabB and FabF proteins, Mtb also possesses KasA and KasB in the F AS II system.

It is proposed that while FabH catalyzes the initial condensation, KasA carries out

an extension to an intermediate stage, followed by an extension to full length

meromycolate by KasB (Bhatt et aI., 2007a; Takayama et aI., 2005).

- 13 -

Chapter 1

1.1.1.5 {3-Ketoacyl-ACP Reductase (FabG)

FabG are tetrameric a/~ proteins that use NADPH as a cofactor and

catalyze the reduction of the ~-ketoacyl-ACP to ~-hydroxyacyl-ACP. These

enzymes belong to the short-chain dehydrogenase/reductase (SDR) superfamily

and possess a classical Rossmann fold for binding of the cofactor in a syn

conformation (Hoang et aI., 2002; Lai and Cronan, 2004). MabA is the homologue

of FabG in the F AS II pathway of Mtb, which is involved in the biosynthesis of

mycolic acids (Takayama et aI., 2005).

1.1.1.6 3(R)-Hydroxyacyl-ACP Dehydratases (FabA and FabZ)

The third step in the elongation cycle is the dehydration of the ~­

hydroxyacyl-ACP generated by FabG to the trans-2-enoyl-ACP. There are two

isozymes that catalyze this reaction: FabA and FabZ. FabA performs a dual

function of dehydration (to form the trans-double bond) and isomerization of the

trans-2- to cis-3-decenoyl-ACP. This step is essential for the formation of

unsaturated fatty acids in E. coli (Birge et aI., 1967; Kass and Bloch, 1967; Kass et

aI., 1967; Silbert and Vagelos, 1967). FabA is always found with the condensase,

FabB, and presence of FabA alone is limited to F AS II systems found in gram­

negative bacteria that produce unsaturated fatty acids (Rosenfeld et aI., 1973).

While FabA catalyzes the essential isomerization reaction necessary to introduce

the cis-double bond, FabB is responsible for elongation of the cis-unsaturated

intermediates (Cronan, 1996; Garwin et aI., 1980). The second dehydratase, FabZ

was discovered as a suppressor of temperature-sensitive mutants in lipid A

biosynthesis and does not catalyze the isomerization reaction (Mohan et aI., 1994).

- 14 -

Chapter 1

FabA and FabZ also differ from each other in having different active site residues:

an aspartate in FabA and glutamate in FabZ.

1.1.1.7 Enoyl reductases: FabIIFabKlFabL

FabI is the target of a number of clinically important anti-infective agents,

such as isoniazid and triclosan (Banerjee et aI., 1994; Heath et aI., 1998; Levy et

aI., 1999). This enzyme uses NADH to reduce the C2-C3 carbon-carbon doubl.e

bond generated by the dehydratase enzymes to complete one round of the

elongation cycle. It is believed that FabI shows specificity for NADH rather than

NADPH due to the absence of a charged pocket for an adenine ribose phosphate of

NADPH to fit in. However, some FabIs, such as the Staphylococcus aureus FabI

use NADPH as a cofactor and are expected to contain this pocket (Heath et aI.,

2000). In the F AS II pathway of Mtb, InhA is the homologue of FabI and is a

target of the frontline tuberculosis drug, isoniazid (Marrakchi;,et aI., 2000). There

are two other isoforms ofFabI found in bacteria: FabK and FabL. The FabK group

is found in gram-positive bacteria and consists of FMN-containing, NADH­

dependent ERs with no similarity to FabI in the primary sequence (Heath et aI.,

2000). FabL shares some degree of sequence similarity with FabI proteins, but not

enough to allow for its classification as a FabI (Heath et aI., 2000).

1.1.2 Type I fatty acid synthases

The F AS I system comprises of all the catalytic domains on a single

polypeptide and catalyzes the iterative condensation of typically seven malonate

units with an acetyl-starter. The acetyl-primer is loaded onto the p-pant arm of

ACP by the AT domain followed by an intra-molecular transfer ofthis starter chain

- 15 -

Chapter 1

to the keto synthase (KS) domain. The malonate-extender unit is then transferred

to the vacant p-pant arm of ACP by the same AT domain, which exhibits a dual

specificity for malonate- and acetate groups (Plate et aI., 1970). This is followed

by a condensation reaction mediated by the KS domain and subsequent processing

, of the ~-carbon by the ketoreductase (KR), dehydratase (DR) and the enoyl-

reductase (ER) domains. Both the KR and the ER domains utilize NADPR as a

hydride donor for the reduction reaction. After completion of one round of

condensation, the saturated intermediate IS transferred from the

phosphopantetheine arm of ACP to the active-site cysteine of the KS, through an

innate transferase reaction of the KS domain. The vacant phosphopantetheine site

is again loaded with another unit of malonyl-CoA which continues the cycle

further for seven times. After this, the sixteen carbon chain is released from the

ACP phosphopantetheine through the action of the chain-terminating TE domain.

The F AS I reaction is pictorially depicted in figure 1.3.

2 3 I os AT DH Eft KR """ TEl KR..:::::r" # k 0< '-,---,.--'

1 ~,. o=< TypelFAS

Figure 1.3: The FAS I biosynthetic pathway of E. coli. The sequence of reactions is indicated by numbersing on the F AS proteins.

------------Since all catalytic sites in case of the F AS I system are present on a single

polypeptide chain, the position of the constituent domains within the F AS

- 16-

Chapter 1

assembly was of interest to a number of researchers. F AS proteins were isolated

from various organisms and were subjected to a number of biochemical studies

including peptide mapping, active-site labelling and limited proteolysis (Hardie,

1986; Smith et aI., 1976; Tsukamoto et aI., 1983). As a result of these studies, it

became clear that the FAS I system can be further divided into two subgroups: 0.54

MDa animal F AS comprising of two identical polypeptides and the fungal F AS

comprising of six pairs of non-identical subunits with a collective mass of 2.6

MDa.

1.1.2.1 Structural models/or animal Type I FAS

Work in several laboratories in the 1970s established the dimeric nature of

animal F AS and revealed that these proteins could be dissociated to their

corresponding monomers by using low ionic strength buffers at low temperatures

(Kumar et aI., 1970; Smith and Abraham, 1971). The monomers were found to be

inactive but the activity could be restored on their reassociation to the dimeric

form. Jim Stoops and Salih Wakil cross-linked the KS of one subunit with the p­

pant arm of ACP from another subunit using a dibromopropanone linker (Wakil

and Stoops, 1983). These results suggested that catalysis required participation of

both the subunits and led to the first model for F AS I enzymes (Figure 1.4).

According to this model, the two subunits are oriented in a head to tail fashion,

such that two sites for condensation are created at the subunit interface. The model

is formed by direct juxtaposition of the KS active site cysteine thiol of one subunit

with the p-pant arm of the ACP from the second subunit. This model of F AS

assembly enjoyed wide acceptance for a long time and could indeed support most

experimental observations reported till that time.

- 17-

Chapter 1

Figure 1.4: Head to tail model for animal FAS I proteins. The two reaction centers are encircled.

The model was put to test by a series of studies involving expression of

recombinant FAS mutants in insect cell lines (Joshi and Smith, 1993). Different

F AS proteins containing mutations in various domains were combined with each

other and subjected to dissociation and reassociation to form a mixed population of

hetero- and homo-dimers. The hetero-dimers were then analyzed for activity

(Witkowski et aI., 1996). The most surprising finding of these studies was that the

ACP domain could functionally interact with the AT and KS domains of any of the

two subunits (Joshi et aI., 1998b). Also, the DR domain was found to interact with

the ACP domain on the same subunit, which was separated by more than 1100

residues (Joshi et aI., 1997). These observations opposed the prevailing head to

tail model, which had predicted that the ACP domain of one subunit cannot make

any functional contacts with the AT, KS and DR domains on the same subunit.

The conventional head to tail model was modified and it was suggested that the

domains do not lie in a flat way but coil in 3-dimensional space, so as to allow

functional interactions between domains distantly located on the same subunit.

Further refinements in the experimental procedures were made by using

differently tagged subunits and obtaining hetero-dimers at a higher purity (Joshi et

- 18 -

Chapter 1

aI., 1998a). This enabled precise assessment of the specific activity of the hetero-

dimers and confirmed the earlier findings (Rangan et aI., 2001). The

dibromopropanone coupling pattern observed for F AS proteins was also

reexamined. Three molecular species could be identified on running the

dibromopropanone treated FAS proteins on a SDS-polyacrylamide gel (Witkowski

et aI., 1999). While two of these species could be accounted ~t6e doubly and

singly linked F AS subunits, the head to tail model failed to explain the formation

of the third species (which was later found to be internally cross-linked KS and

ACP domains of the same subunit). This created further doubts about the

assembly of F AS subunits in a head to tail fashion.

Structural elucidation of the KS domains associated with the F AS II system

suggests that these KS domains are universally dimeric proteins and the substrate

binding pocket comprises of residues from both the subunits (Moche et aI., 1999;

Olsen et aI., 2001; Price et aI., 2003). This was contrary to the head to tail model,

wherein the two KS domains were suggested to lie at the opposite poles of the

dimer. The oligomeric status of the KS domains in a F AS I protein was

investigated by truncating the KS domain from the synthase. It was observed that

these F AS I proteins lacking the KS domain did not form dimers. In another

experiment, a cysteine residue was engineered at the N-terminus of the F AS I

protein and was subjected to cross linking studies with a bis-maleimido linker

(Witkowski et aI., 2004). Up to 98% of the engineered subunits could be cross

linked, suggesting that the two KS domains lie close to each othell"o Proteolytic

digestion of the cross linked subunits followed by mass spectrometric sequencing

completely supported these observations and led to a rejection of the conventional

- 19 -

Chapter 1

head to tail model. A new revised model for F AS I was proposed and was called

the head to head model due to close proximity of the KS domains on the two

subunits (Figurel.5). This model allowed for all intra- and inter-domain contacts

and could explain most of the experimental observations previously reported for

F AS I proteins (Witkowski et aI., 2004).

Figure 1.5: Head to head model for animal FAS I proteins (Witkowski et al., 2004).

1.1.2.2 Electron microscopy and crystallographic studies on anima~ Type I FAS

A number of groups also adopted a more direct approach and used electron

microscopy or X-ray crystallography to understand the assembly ofFAS I proteins.

However, the large size of these proteins and the inherent conformational

variability associated with them posed a big challenge. This was overcome by

using catalytic mutants of F AS and imaging them in presence of reaction substrates

(Asturias et aI., 2005). Under these conditions, the F AS proteins were staDed at an

intermediate stage of catalysis and showed restricted conformational mobility.

Low resolution images (~30A) were obtained for the FAS proteins and revealed

that the structures exhibited pseudosymmetry about an axis running through the

body of the dimer, but at right angles to that proposed in the conventional head-to-

- 20-

Chapter 1

tail model (Figurel.6a). Also, the two halves of the structure were not completely

symmetrical and one side appeared to contain significantly more electron density

than the other.

(a)

(b)

Figure 1.6: (a) Cys161Gln rat FAS mutant imaged under turnover conditions and preserved in amorphous ice. (b) Alternative arrangements of the two subunits that is consistent with the structure of the dimer (Smith and Tsai, 2007).

When the structure was solved for an N-terminally His6-tagged F AS dimer

that had been labelled with a nanogold Ni2+ -nitriloacetic acid complex, two gold

clusters were located near the center of the structure. These findings were

interpreted to indicate that the two F AS monomers coil in an arrangement

consistent with the head-to-head model. However, the reconstructions did not

allow distinction between back-to-back or cross-over subunit arrangements (Figure

l.6b).

Many of the controversial issues regarding the structural organization of

type I F AS proteins were resolved by the crystallization of the complete animal

FAS protein by Nenad Ban's group at Zurich (Figurel.7) (Maier et ai., 2006).

They were successful in obtaining crystals of the full length porcine F AS that

Chapter 1

diffracted at 4.5A, but this resolution \-vas insufficient to identify individual

domains or to trace the complete backbone of the two subunits .

Figure 1.7 : Structura l overview of the type I FAS. The experimental electron dens it~ is in gn:~

and the titted FAS II domains are shol\ n in colour (Smith and Tsai. 2007 ).

In order to resolve this . an interesting approach was adopted wherein high-

resolution structures of individual homologous type II bacterial proteins were titted

into the electron density maps of the full length FAS. This revealed an X-shaped

organization for the F AS protein and confirmed the position of the KS domain in

the central core of the structure. The central body of the structure is composed of

the KS, DH and ER dimers and is tlanked by the monomeric KR and AT domains.

Conspicuous by their absence from the structure were the ACP and TE domains.

for which no appropriate electron density could be identitied. It was suggested th" t

the inherent mobility of these domains was responsible for the lack of any electrc,n

density. The location of these domains was thought to be at the extremity of t' le

KR arms as the ACP and TE domains follows the KR domain in the lint:ar

sequence of F AS. Careful analysis of the structure in fact revealed blurred elect ron

density at the end of one of the arms. Since the complete F AS backbone could not

be traced through the interdomain connecting regions, it was still unclear \\ he lher

the arms and legs on the same side of the structure are associated \\ ith the SJme

- 22 -

Chapter I

subunit or come from opposite subunits of the dimer. It was also deduced from the

structure that the active sites of the two sets of domains are oriented facing each of

the two lateral clefts in the structure and constitute two discrete reaction chamber:, .

However. the two chambers are not identical and one of them was found to be

narrower than the other. Hinge regions that faci I itate conformational tlexi bi I ity ill

F AS were also identitied by superimposing two sides of the F AS structure on eac )

other. Though the structure provided considerable insights into F AS I assembl).

the mode of interactions of ACP with the KS and AT domains of both the subunit-;

was not clear.

Front View

Figure 1.8: Structural overview of the type I FAS at 3.2A. reso lu tio n (Maier et aI., 2008).

The same group managed to improve the resolution of the mammal ian F AS

I structure to 3.2A but the position of the ACP and the TE domains in the structure

still could not be located (Figure 1.8) (Maier et al.. 2008). However. the

connectivity of the domains and detailed features of the act ive sites could be

visualized well. Apart from the lower condensing portion (KS and AT domains)

and the upper ~-carbon modifying portion (OH, ER and KR domains), two

additional nonenzymatic domains were located at the periphery of the mod i fy i ng

- 23 -

Chapter 1

part. While the first domain showed homology to the methyl transferase family

and was called 'pseudo-methyl transferase" ('PME), the other represented a

truncated KR fold and was referred to as "pseudo-ketoreductase" ('PKR). The

structure also revealed that the condensing and the modifYing parts are loosely

connected and form only tangential contacts. This would enable rotation of the

two parts with respect to each other and could contribute to substrate shuttling

across the two reaction chambers. However, alternate connectivities· between the

two parts can exist in another conformation of the F AS protein.

1.2 POLYKETIDE SYNTHASES

Polyketides were first discovered in 1883 when James Collie in London

University was working on the elucidation of structure of dehydroacetic acid

(Collie and Myers, 1893). He observed that boiling dehydroacetic acid with

barium hydroxide yielded an aromatic compound called orcinol as one of the

products. Further investigation revealed a poly ketone intermediate involved in this

conversion. Based on his findings, he proposed that simple condensation of acetyl­

groups could produce a number of pyridine, benzene, and naphthalene derivatives

(Collie, 1893). He coined the term polyketide (i.e., polyacetate) for the compounds

containing the structure CH3-CO-(CH2-COkX and suggested a complex series of

reactions for the conversion of polyketones to their final form (Collie, ] 907).

However, his theories were majorly neglected and the polyketide field saw much

less advancement during that time.

Like in the case of fatty acid biosynthesis, main impetus to the polyketide

field came with the availability of isotopically-labelled compounds. Arthur Birch

in the 1950s recognized that polyketones could be generated from condensation of

- 24-

Chapter 1

acetate units and decided to test this hypothesis (Birch et al., 1955; Shoolingin­

Jordan and Campuzano, 1999). He performed radioactive feeding experiments,

wherein Penicillium patulum, known to produce the aromatic polyketide, 6-

methylsalicylic acid (6-MSA) was fed with 14C acetate labelled at the first carbon.

Birch worked out a detailed biosynthesis of 6-MSA from acetate units and

proposed that four sites in 6-MSA should incorporate the radiolabel. The results of

his experiments were consistent with Birch's prediction and led to a series of

studies wherein many organisms known to produce secondary metabolites were

subjected to similar analysis. These experiments established that many

polyphenolic aromatic molecules are biosynthesized from acetate units, according

to what came to be known as the 'Collie-Birch polyketide hypothesis'. They also

revealed that many non-aromatic compounds were formed by further

transformations of such products. Another notable contribution towards the

polyketide field was made by Tom Harris in 1970s when he reported that the in

vivo folding of a polyketone chain is under enzymatic control and unwante:d

cyc1ization modes are totally suppressed. In particular, premature cyc1ization of

the chain doesn't occur during early stages of elongation (Harris and Wittek,

1975).

In 1984, with the advancement of genetic techniques, the first set of genes

encoding for biosynthesis of an aromatic polyketide, actinorhodin, were identified

in David Hopwood's laboratory (Malpartida and Hopwood, 1984). The genes

were sequenced and the primary sequence of various proteins was established.

Interestingly, a number of proteins showed convincing homology with enzymes

belonging to the fatty acid synthase family (KS, ACP, and KR). The genes were

- 25 -

Chapter 1

discrete and the actinorhodin PKS was proposed to be a type II dissociable system

like the F AS II system of bacteria. This was followed by the discovenj and

cloning of the genes encoding the enzymes for erythromycin biosynthesis in

Saccharopolyspora erythraea (Formerly Streptomyces erythraeus) by the groups

led by Peter Leadlay and Leonard Katz (Cortes et aI., 1990; Tuan et aI., 1990).

The genes encoding the biosynthetic enzymes for the aglycone portion of this

antibiotic were found in three long open reading frames, each coding for an

enormous polypeptide of ~350 kDa. Remarkably, the amino acid sequence of the

encoded proteins strikingly resembled the type I animal F AS proteins. The three

polypeptides together were predicted to contain six sets of catalytic sites or

modules. Some of these modules lacked certain enzymes responsible for p-carbon

processing, and only the last module possessed an enzymatic domain resembling a

chain-terminating thioesterase (TE). Interestingly, the chemical structure of the

polyketide product had a direct correlation with the genetic organization of the

modules, and unlike F AS, each module was expected to catalyze only one round of

condensation and pass the extended chain from the ACP of one module to the KS

of the next (Bevitt et aI., 1992; Donadio and Katz, 1992; Donadio et aI., 1991).

Cell free reconstitution of these proteins and their genetic engineering (~nabled the

study of PKSs in great details and built the foundation of the discovery of many

new macrolides as well as mechanistic themes for polyketide condensation

(Jacobsen et aI., 1997).

It was recognized that PKSs in general possess two set of catalytic sites or

domains: core domains and the auxiliary domains (Gokhale, 2001). The core

domains comprise the KS, AT and ACP domains and are responsible for the

- 26-

Chapter 1

decarboxylative Claisen type condensation of the ketide units. The auxiliary

domains mayor may not be present in a given PKS and are responsible for the ~.-

carbon modification. Majority of the PKS enzymes contain combinations of the

KR, DH or the ER auxiliary domains. However, there are examples wherein

domains like methyl transferases, acyl-CoA ligases or thioesterases may be present

as a part of the PKS architecture (Aron et aI., 2005; Harvey et aI., 2006; Haydock

et aI., 2004; Miller et aI., 2002). The flexibility of a given PKS in having these

auxiliary domains leads to a lot of structural variability in the polyketide backbone,

which is further augmented by the broad substrate specificity of these enzymes

(Gokhale et aI., 2007a). Unlike FASs, the PKS enzymes can utilize a range of

starter and extender units, the most common extender units being malonyl-CoA

and methylmalonyl-CoA (Hopwood, 1997; Katz and Dopadio, 1993). A

comparative analysis of the F AS and PKS enzymes and the functional significance

of various catalytic domains is shown in Figure l.9.

STARTER UNIT EXTENDER UNIT

1 18 o 0 0

R)lS-E) HO¥s-e ~ R'

co.-1 EJ o °

R¥S-Ac3 ~ OH 0 R' 'V ~s RJ...J(g-t'CP'l

Polyketidei /v:..~ '?, - '( 'C.I FAS intermediat'kR o:;\. 0 R"

~~~ R~ Jl~'CPI o - ....... , - 'C.I~.t>

Y R' .,;a\u<;G' R ~ ~1'<>"

R' ~u(O~ 9<o6u

, Reduced polyketides R=H,CH,

Figure 1.9: Comparison ofPKS and FAS catalysis.

- 27 -

Chapter 1

Based on the structural and functional understanding of PKS proteins and

their close homology to F AS enzymes, PKSs are architecturally classified as type

I, II and III enzymes. The type I PKSs resemble the yeast and animal F AS and

contain all the active sites on a single polypeptide chain. These can be further

classified into two subgroups. Type I modular enzymes contain a specific set of

catalytic sites for each round of chain elongation and are present as a large PKS

cluster containing multiple modular PKSs (Caffrey et al., 1992). The reduction

chemistry of each ketide unit is dictated by the domain organization of the

respective PKS enzyme. On the other hand, type I iterative enzymes use the same

set of catalytic sites in a repetitive manner and append the same type of ketide unit

with each extension cycle (Bentley and Bennett, 1999; O'Hagan, 1993). In type II

PKSs, each active site is present on a separate polypeptide chain. The complete set

of discrete mono-functional enzymes form a multienzyme complex and the active

sites are used iteratively to catalyze various condensation and chain elongation

steps (Bibb et al., 1989; Fernandez-Moreno et al., 1992; Grimm et al., 1994). Type

III or chalcone-synthase like PKS enzymes perform decarboxylation,

condensation, and cyclization using a single active site and are classified amongst

PKS enzymes due to their mechanistic similarity of decarboxylative C1aisen type

condensation. These enzymes use free CoA substrates and do not require the ACP

domain for catalysis (Austin and Noel, 2003; Shen and Hutchinson, 1993; Tropf et

al., 1995).

1.2.1 Type I modular PKSs

Type I modular PKSs contain multiple active sites or enzymatic domains

organized into a module on a single polypeptide chain. Biosynthesis of the

• - 28-

Chapter 1

polyketide chain requires participation of multiple PKS modules and each module

is utilized just once during the entire catalytic cycle. Formation of a carbon-carbon

bond proceeds via the decarboxylative condensation of a ketide extender unit with

the starter chain. This condensation event is followed by a programmed reductive

cycle where the extent of reduction is dictated by the domain organization of the

participating module (Rawlings, 2001a; Rawlings, 200Ib). The modular PKS

clusters often contain a loading module at the front of the first module (that is

responsible for obtaining the starter unit) and a thioesterase at the end of the last

module (that is responsible for unloading the final product). The PKS enzymes

responsible for the biosynthesis of 6-deoxyerythronolide B (6-DEB), the aglycone

portion of erythromycin A are a classical example of modular synthases and have

been studied in great details (Cortes et aI., 1990; Donadio et ai., 1991; Rawlings,

2001a).

6-DEB is biosynthesized in S. erythraea by condensation of propionate and

methylmalonate units. After its assembly, it undergoes oxidation by the action of

cytochrome P-450s, followed by glycosylation to yield erythromycin A. Three

independent proteins, DEBSI, DEBS2 and DEBS3 are involved in the biosynthesis

of 6-DEB and encompass 35 kb of DNA (Figure 1.10) (Donadio et aI., 1991).

Each protein was found t~ contain two modules in addition to a loading module at

the N-terminus and a chain terminating thioesterase domain at the C-terminus of

module 6 in DEBS3. The loading module contains the AT and ACP domains and ,

loads propionyl-CoA onto the first module, which catalyzes its condensation with a

methylmalonate unit to form a diketide. Each module then adds a methylmalonate

unit and processes it according to the auxiliary domains present, to synthesize the

- 29-

Chapter 1

heptaketide product. Five of the six DEBS modules carry active KR domains, with

only DEBS module 3 harboring a KR like but catalytically inactive, domain

(Khosla et ai., 2007). At the end of the sixth module, the TE domain cyclizes and

releases the ketide chain as 6-DEB (Kao et ai., 1994). The domain organization of

the DEBS cluster and the reactions catalyzed by each PKS is shown in Figure 1.10.

~ ____ ~~,_" ____ ~)I~ __ ~~~AI_' __ ~>~I ____ ~~_.'_" __ ~> n

-.,.>-

Figure 1.10: Domain organization of the erythromycin polyketide synthase and biosynthesis of6-DEB (Staunton and Weissman, 2001).

Since the discovery of the genes involved in biosynthesis of erythromycin,

many other genes encoding for macrolide PKSs have been sequenced and include

those involved in the biosynthesis of rapamycin (Aparicio et aI., 1996; Schwecke

et aI., 1995), FK506 (Motamedi et aI., 1997; Motamedi and Shafiee, 1998),

spiramycin (Kuhstoss et aI., 1996), nystatin (Brautaset et aI., 2000; Zotchev et aI.,

2000), tylosin (Gandecha et aI., 1997) etc. Each PKS involved in the biosynthesis

of these compounds has a modular organization analogous to DEBS proteins and

catalyzes a single condensation and reduction cycle. However, there are examples

- 30-

Chapter 1

wherein a deviation from this modular logic has been reported (Haynes and

Challis, 2007). For instance, iterative use of modules has been reported for PKSs

involved in the biosynthesis of borrelidin, aureothin, stigmatellin, lankacidin and

some minor compounds isolated from Sac. Erythraea (He and Hertweck, 2003; He

and Hertweck, 2005; Olano et ai., 2003; Wilkinson et ai., 2000). These PKS

systems use one of the modules of the modular assembly line in an iterative

fashion to catalyze two or three rounds of chain extension as a programmed event

in the biosynthesis of the polyketide product. It is not yet possible to determine

whether a module will act iteratively or in a modular fashion from sequence

comparisons alone. Another deviation from the modular PKS logic is the skipping

of modules in an assembly line. This has been observed in case of PKSs involved

in the biosynthesis of methymycin, pikromycin, and some engineered constructs of

the DEBS system (Aldrich et ai., 2005; Moss et ai., 2004; Rowe et ai., 2001).

Another interesting feature of modular PKS systems is seen in the case of

PKS clusters devoid of AT domains. For a long time, these clusters were believed

to be non-functional, as the AT domain is a core component of a PKS protein and

is essential for activity. Recent studies have revealed that these 'AT -less' PKS

systems involve the iterative use of one or two external AT domains to load

extender units onto their ACP domains (Cheng et ai., 2003). The AT domain acts

in trans and this phenomenon has now been reported in a number of PKS systems

like those involved in the biosynthesis of.1einamycin (Tang et ai., 2004), lankacidin

(Arakawa et ai., 2005), chivosazol (Perl ova et ai., 2006), and disorazol (Kopp et

ai., 2005). Modular PKSs also show a great deal of versatility by forming

functional links with non ribosomal peptide synthatases (NRPSs) and catalyzing

- 31 -

Chapter 1

the biosynthesis of hybrid polyketide-polypeptide compounds (Admiraal et aI.,

2003; Aparicio et aI., 1996; Gehring et aI., 1998; Pelludat et aI., 1998).

1.2.1.1 Inter-protein docking complexes

Specific transfer of the intermediate chains between various PKS modules

is crucial for the correct assembly of the final polyketide metabolite and to avoid

wasteful usage of the host biosynthetic machinery. Transfer to the wrong module

can lead to complete abrogation of synthesis due to stalling of misprimed PKSs or

lead to the production of a dysfunctional compound. The specificity of

communication between modules is thus important and is maintained in case of

bimodular proteins as the transfer occurs between covalently linked modules. On

the other hand, the specificity of transfer between modules present on separate

polypeptides is maintained by formation of a docking complex between the C­

terminus linker of the donor module.,.and the N-terminus linker of the acceptor

module (Broadhurst et aI., 2003; Gokhale et aI., 1999b). These docking complexes

ensure that each polypeptide interacts only with its appropriate partner in the

assembly line and functions independent of the catalytic domains to which they are

attached. These domains can be easily exploited to facilitate inter-modular chain

transfer between unnatural polypeptide partners and can be used to engineer novel

compounds.

An insight into this recognition mechanism was provided by the NMR

solution structure of a l20-residue fusion protein consisting of the docking domain

of DEBS2 and DEBS3 (Figure 1.11) (Broadhurst et aI., 2003). The structure

revealed that the docking complex adopts a stable dimeric structure in which

residues 1-80 from the C-terminus of donor module contribute 3 a-helices and

- 32-

Chapter 1

residues 83-120 from the N-terminus of the acceptor module contrihutes a single

helix. Interactions between helix)/2 from the C-terminus and helix 3/4 from the ,. ~()U-I

C- and N-terminus, respectively, divide the structure into two subdomains, A and

B, which are connected by flexible tethers that do not interact with the two

subdomains.

Donor module

_ Subdomain A

_ Sutxfomain B

Acceptor module Figure 1.11: NMR solution structure of docking complex between DEBS 2/3 (Khosla et al., 2007).

Sub domain A contains an unusual intertwined four a-helix bundle formed

by helices 1, 2, l' and 2' and is believed to be involved in the stabilization of the

entire DEBS2 dimer at its C-terminus. Subdomain B is composed of helices 3, 3',

4 and 4' and forms a parallel coiled-coil dimer, which may also play a role in

stabilization of the DEBS3 dimer at its N-terminus. Since subdomain B contains

residues from both the modules, it is the one believed to determine the specificity

of transfer between modules. In fact, salt bridges and hydrophobic connections

between helices from the two modules can be identified in the dockhlg complex.

Site directed mutagenesis of these residues leads to drastic changes in overall

catalysis, suggesting their importance in mediating inter-modular communication

- 33 -

Chapter 1

between modules present on separate polypeptides (Chopra et aI., 2008; Weissman,

2006).

1.2.2 Type I iterative PKSs

Type I iterative PKSs are the enzymes that catalyze F AS like iterative

condensation of the same kind of ketide unit in a repetitive fashion (Bentley and

Bennett, 1999; O'Hagan, 1993). The classical examples of these enzymes are the

fungal metabolites 6-MSA, aflatoxin, and lovastatin (also called mevinolin or

monaco lin K) (Hendrickson et aI., 1999; Shoolingin-Jordan and Campuzano,

1999). The enzyme involved in the biosynthesis of 6-MSA contains the KS, MAT,

DH, KR and ACP domains and has been purified from Penicillium patalum (Fujii

et aI., 1996). These activities act repeatedly to catalyze the condensation of one

acetate and three malonate molecules, carrying out different levels of reductive:

processing at every stage. The molecular basis of this kind of programming in case

of iterative PKSs is not very well understood. The tetra-ketide thus formed, is

cyclized via an intramolecular aldol condensation and aromatization to yield the

final metabolite (Beck et aI., 1990; Bentley and Bennett, 1999; Staunton and

Weissman, 2001).

Another well documented example of an iterative type I PKS is lovastatin

synthase (Hutchinson et aI., 2000). Lovastatin is produced by the filamentous

fungus Aspergillus terre us and inhibits the reductase responsible for the conversion

of (3S)-hydromethylglutaryl-CoA (HMG-CoA) into mevalonate during cholesterol

biosynthesis. Lovastatin therefore exhibits strong cholesterol lowering activity and

is used clinically to reduce serum cholesterol levels (Alberts et aI., 1980). The

chemical structure of lovastatin is composed of two polyketide chains joined

- 34-

Chapter 1

through an ester linkage (Figure 1.12). One chain is a nonaketide that undergoes

cyclization to form an octahydronaphthalene ring system, and the other is a

diketide, (2R)-2-methylbutyrate. The lovastatin biosynthetic cluster spans a region

of 64 kb and includes 18 genes. Four of these genes, 10 vB , 10vF, 10vC and 10vD

have been shown to be essential for the biosynthesis of lovastatin. 10vB codes for

the nonaketide synthase and 10vF for the diketide synthase. love and 10vD code

for an ER and a transesterase, respectively (Staunton and Weissman, 2001).

CH,eos.coA +

Ctt.COs.ccA I COOH

Dlhydromonacolln L Monaco!!n L MonacoHnJ

Figure 1.12: Proposed intermediate steps in the biosynthesis of lovastatin .

CH,c05-CoA +

CH,cos.ccA I CQOH

Apart from containing a full set of catalytic domains required for complete

saturation of the ketide chain, the nonaketide synthase also contains two additional

domains: a methyl transferase domain positioned between the DR and the ER

domains and a C-terminal domain homologous to the condensation domains of

NRPSs. Biochemical reconstitution of this protein revealed that LovB could

catalyze the iterative biosynthesis of a hexaketide and a heptaketide product, but

with a lower degree of expected reduction (Kennedy et ai., 1999). Interestingly,

this aberrant behaviour could be rescued by the lovC gene which encodes an ER

domain and led to the synthesis of monacolin J. The second PKS gene, 10vF, was

- 35 -

Chapter 1

identified to be a non-iterative PKS and is involved in the biosynthesis of the 2-

methylbutyrate side chain of lovastatin. The domain organization of this protein is

identical to the nonaketide synthase except that it possessed an active ER domain

and lacked the carboxy terminal condensation domain. The lovD gene was

suggested to be a transesterase involved in the synthesis of mature lovastatin by

catalyzing the final esterification of the independently synthesized diketide to the

nonaketide (Kennedy et aI., 1999).

1.2.3 Type II PKSs

Type II PKSs form a multi enzyme complex similar to the F AS II systems

found in plants and bacteria and consist of discrete mono-functional enzymes that

act iteratively to produce the polyketide metabolite (Carreras and Khosla, 1998;

Hopwood, 1997). These PKSs contain a single set of active sites carried on

separate proteins namely KS, chain length factor (CLF), ACP and a malonyl

CoA:ACP transacy lase (MAT) (Bao et aI., 1998; Carreras and Khosla, 1998). This

assembly constitutes the minimal PKS responsible for generating the carbon

backbone of the polyketide. The cyclization, aromatization and various

oxidation/reduction reactions are catalyzed by a host of additional discrete

enzymes. The KS-CLF heterodimeric complex catalyzes the chain initiation and

iterative elongation of the polyketide backbone. Analysis of the CLF domain has

revealed that the catalytic cysteine is replaced by a glutamine in these domains and

this mutation converts them into efficient decarboxylases (Gokhale, 2001). The

crystal structure of the KS-CLF heterodimer has been solved and an amphipathic

tunnel approximately 17 A in length at the heterodimer interface explains the

structural control of chain length by the CLF (Keatinge-Clay et aI., 2004). The

- 36-

Chapter 1

MAT protein is responsible for the transfer of malonate units from malonyl CoA to

the p-pant arm of ACP, which participates in the condensation reaction with the

KS-CLF heterodimer. The MAT domain is shared between the type II PKS and

the housekeeping FAS proteins (Carreras and Khosla, 1998).

Figure 1.13: Reaction scheme ofa type II PKS (Gokhale, 2001).

Two well studied examples of a type II PKS system are the actinorhodin

synthase and the tetracenomycin synthase, involved in the biosynthesis of aromatic

polyketides. While the actinorhodin synthase utilizes 8 malonyl-CoA units to form

the 16-carbon chain, the tetracenomycin synthase catalyzes the biosynthesis of a

20-carbon chain from 10 malonyl-CoA units (Gokhale, 2001). A model depicting

the association of individual domains for the synthesis of aromatic polyketide

actinorhodin is shown in Figure 1.13.

1.2.4 Type III PKSs

Type III PKSs differ from their type I and type II counterparts in having a

homodimeric structure that performs iterative condensation of free CoA thioesters,

without the involvement of ACP (Austin and Noel, 2003; Jez et aI., 2001; Lanz et

aI., 1991; Shen and Hutchinson, 1993; Tropf et aI., 1995). These enzymes are

grouped into the PKS superfamily due to their mechanistic similarity of

decarboxylative 2-carbon condensation. Classically, they have been characterized

- 37-

Chapter 1 --------------------------------------------------------

from the plant kingdom where they catalyze the biosynthesis of chalcones, which

act as precursors to a number of plant metabolites (Schroder and Schroder, 1990).

As a result, the plant type III PKS enzymes are referred to as chalcone synthases

(CHS). Genome sequencing projects of various microbes in recent years has

revealed their presence in organisms like S. coelicolor, Bacillus subtilis, B.

halodurans, Deinococcus radiodurans, Cytophaga hutchinsonii and Mtb. The

functional role of these enzymes in these organisms is not very clear. A recent

report suggested that type III PKSs are involved in the biosynthesis of long-chain

alkyl resorcinols and alkyl pyrones in Azotobacter vinelandii. Similar compounds

were identified as major chemical components of the protective cyst coat of the

bacterium, which confers resistance to various chemical and physical agents during

dormancy (Funa et aI., 2006). Interestingly, both resorcinols as well as pyrones

can be formed from the same tetra-ketide intermediate by two different

mechanisms of cyclization.

In plants, CHSs are involved in the biosynthesis of compounds that play an

important role in processes like antimicrobial defense, flower pigmentation, pollen

fertility etc. These enzymes typically use 4-coumaroyl CoA as a starter substrate

and perform up to three decarboxylative condensations with malonyl-CoA to

synthesize the linear polyketide intermediates. Subsequent cyclization of the linear

intermediate within the active site cavity of these enzymes results in the final

product (Austin and Noel, 2003; Jez et aI., 2001). Acridone synthase (Baumert et

aI., 1994a; Baumert et aI., I 994b), 2-pyrone synthase (2-PS) (Helariutta et ail.,

1995), benzalacetone synthase (Borejsza-Wysocki and Hrazdina, 1996), bibenzyl

synthase (Preisig-Muller et aI., 1995), homoeriodictyol synthase (Christensen et

- 38-

Chapter 1

al.. 1998) and benzophenone synthase (Schmidt and Beerhues. 1997) are other

members of this superfami Iy. They differ from the chalcone synthases in uti I izi ng

non-phenyl isoprenoid starters or carrying out different number of condensation ~ , .

Some plant enzy mes also show different cyclization pattern s emphasizing th e

growing diversi ty of these enzymes.

The firs t crystal structure of a type III PKS was of a CHS from alfalfa and

revealed a two fold axis of symmetry runn ing through a symmetric protein dilT,er

(Figure 1.14) (Ferrer et aI., 1999). The homodimers were found to contain t,,\ 0

distinct and functionally independent bilobed active site cavities. situated at Ihe

bottom of the conserved core in each monomer. One lobe form s a coum"r),1

binding pocket and the other accommodates the growing polyketide chain be o re

cyclization occurs . Four residues, C)'s 164. Phe215. His303 and Asn336 form the

active site of the CHS and are conserved in all CHS-related enzymes. Compar ison

of the structures of CHS and 2-PS revealed that the 2-PS active site is

approximate ly one third the size of the CHS cavity, implying that the VolLII' le 01"

the active site cavity influences starter molecule selectivity and limits polyketide

length between the two PKSs enzymes (Ferrer et aI., 1999; Jez et a l.. 200 I ).

(a) (b)

Figure 1.14: Th ree dimensional structures of type III PKS proteins . (a) CHS2 from Medicago sativa. (b) PKSI8 from Mtb (Ferrer et aI., 1999; Sankaranarayanan et aI., 200~ ) .

- 39 -

Chapter 1

Type III PKS involved in the biosynthesis of 1, 3, 6, 8-tetra-hydroxyl

naphthalene (THN) has also been characterized from four different species of

Streptomyces (Funa et al., 1999). All these THN synthases use malonyl CoA as

both starter and extender units and synthesize the pentaketide chain, which is

cyclized to THN. The THN synthase from S. grise us (also known as RppA) has

been shown to possess broad starter substrate specificity and synthesizes a wide

variety of products (Funa et al., 2002a; Funa et al., 2002b). Similar CHS-like

protein called PhlD was reported to be involved in the synthesis of 2, 4-

diacetylphloroglucinol (DAPG) in Pseudomonas fluorescens using acetoacetyl-

CoA as a starter unit (Bangera and Thomashow, 1999). Type III PKS proteins

have also been found in Mtb (Gokhale et al., 2007b; Sankaranarayanan et al., 2004;

Saxena et al., 2003). The in vitro reconstitution of the mycobacterial PKS18

protein reveals that this protein does not utilize the plant-specific acyl-CoA ./

substrates but catalyzes the synthesis of long-chain tri-ketide and tetra-ketide

pyrones using CJ2 to C20 fatty acids as starters. Such unusual starter specificity is

unprecedented in the chalcone synthase family of proteins. The crystallographic

studies with PKS18 protein shows that the overall structure is similar to the CHS

protein but a long hydrophobic tunnel that could accommodate long-chain fatty

acid molecules can be seen in the structure (Figure 1.14) (Sankaranarayanan et al.,

2004).

1.2.5 The PKS domains

1.2.5.1 p-Ketoacyl synthase (KS)

The KS domains catalyze the decarboxylative Claisen-type condensation of

starter acyl chains (thioesterified on the catalytic cysteine residue) with extender

- 40-

Chapter 1

units (typically malonate or methylmalonate) present on ACP, leading to the

formation of ~-ketoacyl-ACP (Heath and Rock, 2002). Apart from catalyzing the

carbon-carbon bond formation, sequence variants of KS domains are known to

catalyze a variety of reactions. The KS domains act like decarboxylases when the

active site cysteine is replaced with glutamine and such KSQ domains appear in the

loading modules for a number of macrolide and aromatic PKSs (Bisang et ai.,

1999). The epithilone PKS has tyrosine instead of the cysteine in active site of the

loading KS (KS Y). Amphotericin, nystatin and PIMSO proteins have serine in

their active site (KSs) and lack both the condensation and decarboxylation

activities (Caffrey et ai., 2001). The KS domains associated with two AT domains

in the loading module are known to condense two substrates instead of one (Ligon

et ai., 2002) and KS domains involved in hybrid NRPSIPKS structures condense

an acyl group onto an amino acid chain. The available crystal structures of

actinorhodin KS-CLF and structures of homologous KAS enzymes reveal that the

KS domains are likely to adopt the thiolase fold (Keatinge-Clay et aI., 2004).

These enzymes are dimers with a large interface of approximately 2400A. Each

subunit has two mixed 5-stranded ~-sheets surrounded by a-helices packed into a

conserved a-~-a-~-a layered fold architecture that comprises of an internally

duplicated helix-sheet-helix motif.

1.2.5.2 Acyl transferase (AT)

The AT domains carry out the critical roles of both initiating polyketide

biosynthesis by selection of starter unit and enabling chain extension by loading

extender units onto ACPs (Liou et ai., 2003). These domains catalyze the transfer

of the substrate moiety from the respective CoA thioester onto the thiol group of

- 41 -

Chapter 1

the p-pant arm of the ACP. Acyl transferases usually exhibit a high degree of

specificity towards specific starter/extender groups (Oliynyk et aI., 1996).

Structural studies on AT domains reveal a unique structural fold in these domains

even though they have an a/~ architecture (Keatinge-Clay et aI., 2003:, Serre et aI.,

1995). The active site contains a typical nucleophilic elbow in a fashion similar to

various serine hydrolases but the catalytic triad is composed of Ser-His-Gln rather

than the usual Ser-His-Asp residues. The specificity of the AT domain of a given

module for malonate or methylmalonate units can be unambiguously predicted by

sequence analysis (Yadav et ai., 2003a). In fact, it is possible to reengineer the

extender unit specificity of a PKS protein by identification and mutational analysis

of the substrate determining residues in the AT domain (Reeves et aI., 2001;

Trivedi et ai., 2005).

1.2.5.3 Acyl carrier protein (ACP)

The ACP is the smallest domain in the PKS module with an approximate

molecular mass of 10 kDa. These domains play an integral role in all the catalytic

steps involved in polyketide biosynthesis. It facilitates intermediate channeling

between vari.ous domains by anchoring the growing chain. This domain is

inactive in its apo-form and is activated after post translational modification of the

catalytic Ser by a 4' p-pant transferase, which adds a p-pant prosthetic group onto

the Ser residue (Walsh et ai., 1997). The intermediate chains are 1hioesterified to

the sulfhydryl group of the 4' p-pant arm of the ACP proteins. The X-ray crystal

structure of the holo-ACP in complex with a trimeric p-pant transferase (Parris et

ai., 2000) and also a few NMR structures .of standalone ACPs (Crump et aI., 1997)

have revealed structural details of the ACP domains. The ACP domain is

- 42-

Chapter 1

composed principally of three major helices and a short helix, with a large loop

region separating helices one and two. The ACP active site consensus sequence is

LGXDSL (Stachelhaus et ai., 1996), which contain the serine where the 4' p-pant

arm is attached (Corteset ai., 1990). However, several ACP domains show

deviations from this signature motif (Aparicio et ai., 2000). In addition to

sequestering the polyketide chain, the ACP also protects the chain from enolization

and premature cyclization.

1.2.5.4 Ketoreductase (KR)

The KR domain is one of the auxiliary domains present in a PKS enzyme

and carries out the NADPH mediated regiospecific ketoreduction of the ~-carbonyl

functionality to a hydroxyl group (Cane et ai., 1998). The stereochemistry of

reduction is an inherent property of the KR domain and this can be exploited to

reprogram a PKS enzyme to yield different stereochemical products (Kao et ai.,

1994). The stereoselective signature motifs for modular PKS proteins are LDD

and PXXXN (Caffrey, 2003; Reid et ai., 2003). It has been proposed that these

motifs control the orientation of the substrate so that the enzyme attaches the

hydroxyl group in the R- configuration. In KR domains that lead to formation of

products with S-configuration, the absence of these motifs combined with the

presence of a characteristic Trp at position 141 dictates the S- stereochemistry.

The sequence comparison of type II aromatic KRs does not reveal any

motif that could be correlated to the stereochemistry of reduction. Based on

structural studies on these KR domains, it has been proposed that the polyketide

chain can bind from the same face as the NADPH pocket (front side motif) or from

the opposite face (back side motif) resulting in an orientation of hydroxyl group

- 43 -

Chapter 1

either in the R- configuration or the S- configuration respectively. These enzymes

have Tyr in the active site within the center of a Rossmann fold (Hadfield et aI.,

2004; Korman et aI., 2004).

1.2.5.5 Dehydratase (DH)

The DH functions after ketoreduction and catalyzes the modification of the

J3-hydroxyl group of the first ketide carbon to an alkene moiety, with the

concomitant release of a water molecule. Sequence comparison of various

catalytically active DH domains from F AS led to the identification of a consensus

motif that was implicated as the signature motif for a functional DH domain

(Bevitt et aI., 1992). Similar signature motifs are also found in PKS DH domains

(Ikeda et aI., 1999). However, subsequent analysis indicated that this motifis also

present in presumably non-functional DH domains. For instance, the structure of

amphotericin suggests that in the biosynthetic cluster, neither of the DH domains

of module 15 and 17 is functional, although the domains contain the conserved

HX3GX3P motif (Caffrey et aI., 2001). It was proposed that inter-domain linker

regions constrain the domain movements in such a way that access to substrate

may be denied even if the domain is otherwise functional. A more detailed

analysis needs to be done for precise identification of residues responsible for

control of catalytic activity of DH domains.

1.2.5.6 Enoyl reductase (ER)

ER domain catalyzes the reduction of the alkene group to an alkane moiety.

Structurally, these domains belong to the NAD(P) binding Rossman-fold liJ<,e

enzymes, having a three layered a-~-a sandwich architecture. A sequence

- 44-

Chapter 1

comparison of niddamycin, erythromycin, and rapamycin ER domains revealed the

presence of a pattern LXHXG(A)XGGVG, which is hypothesized to be the

NAPDH binding motif, and has subsequently been verified by a number of

research groups (Amy et aI., 1989; Scrutton et aI., 1990; Witkowski et aI., 1991).

Domain inactivation and modification experiments on the epothilone PKS from M.

xanthus were found to result in an unexpected 13-oxo derivative, which suggests

another role for these ER domains (Tang et aI., 2005). A recent study on ER

domains from modular PKSs that use methylmalonate extender units revealed a

correlation between a unique Tyr residue in the ER domains and the chirality of the

methyl branch that is introduced (Kwan et aI., 2008). When this position in the

active site is occupied by a Tyr residue, the methyl branch has S- configuration,

otherwise it has R- configuration. However, experiments indicate that additional

residues also participate in determining the stereochemistry of enoylreduction

(Kwan et aI., 2008).

1.2.5.7 Thioesterase (TE)

The TE domains catalyze the cyclization and concomitant release of highly

functionalized polyketides or non-ribosomal peptides via lactonisation (DEBS and

pikromycin), hydrolysis (vancomycin) or lactamization (bacitracin) (Keating et aI.,

2001). Generally, this domain is covalently linked to the ACP of the last

PKSINRPS module but there are reports wherein the chain is released by other

alternate mechanisms.. Prior to cyclization and release, the acyclic polyketide

substrate is transferred from the p-pant arm of the ACP to the TE domain. This

involves the formation of an acyl-O-enzyme intermediate at a conserved Sev·

residue in the active site of the TE domain. The acyl-O-TE intermediate formed by

- 45 -

Chapter .I

acyl transfer from the upstream donor either undergoes hydrolysis or is directed to

an intra-molecular capture by a nucleophilic group in the acyl chain, producing the

cyclic macrolactone (Bruner et aI., 2002). Different TE domains appear to have

different specificity profiles for acyl chains with alternative functionality and

stereochemistry (Gokhale et aI., 1999a; Rowe et aI., 2001). The PKS TEs adopt a

dimeric structure with a hydrophobic leucine rich dimer interface and a substrate

channel that passes through the entire protein.

For numerous hybrid and non-hybrid modular polyketides like bleomycin,

FK520, megalomycin, pikromycin and rifamycin, there are reports of existence of

physically separated mono-functional TE proteins in their biosynthetic cluster.

These standalone TE domains are believed to play an editing role in these clusters

where they hydrolyze aberrant chains from stalled PKSINRPS enzymes (Kim et

aI., 2002; Zhou et aI., 2008). These proteins have an overall tertiary structure of

the a/~ hydrolase family, which includes enzymes such as lipases, esterases and

peptidases.

1.2.6 Structural analysis of PKSs

Like F AS enzymes, mutant complementation was one of the first strategies

exploited for probing details of the functionality of modular PKSs. The results of

these studies revealed a great deal of similarity with F AS enzymes (Gokhale et aI.,

1998; Kao et aI., 1996). As a result, the first model for type I modular PKSs was

based on the classical fully extended, head-to-tail oriented subunit theme (Kao et

aI., 1996). However, proteolytic studies on DEBS 1, 2 and 3 proteins by Peter

Leadlay and Jin Staunton's group at Cambridge questioned the validity of this

model (Staunton et aI., 1996).

- 46-

Chapter J

The Complete DEBS Helical Stack

DEBS l

OE BS2

DEBS3

Figure 1.15: The Cambridge double helix model (Smith and Tsai, 2007).

The DEBS proteins were subjected to proteolytic digestion with a number

of proteases and the fragments thus generated were analyzed by N-tenninal

sequencing. The ol igomeric state of these fragments was also analyzed by gel

filtration studies. The surprising finding of these experiments was the isolation of

TE dimers. which in case of F AS proteolytic studies separate as monomeric

domains. The head to tail model for PKS proteins did not allow the TE domains to

interact and thus the 'Cambridge Model' for PKSs was proposed in 1996 (Figure

1.15) (Aparicio et al.. 1994 : Staunton et al.. 1996: Staunton and Weissman, 200 I).

According to this model. each PKS module pair f0I1115 a dimer with th e

polypeptides oriented head-to-head. rather than the head-to-tail fashion. It \\' ( IS

suggested that the subunits are twisted together to form a helix in which the KS.

AT and ACP domains are positioned at the core of the helix and the optional (3-

carbon processing domains form loops that protrude out from the axis of the hel ix.

The twi st in the helix was proposed to facilitate functional interactions between the

KS and the ACP domains across the subunit interface. thus accounting for the

mutant complementation experiments. At the C-terminus of the last module. the

- 47 -

Chapter 1

hel ical arrangement brings the two TE domains into close proximity. consistent

wi th the proteolytic studies.

One ambiguity in the Cambridge model, however, was with the stability of

the dimeric modules . Although the modules were found to be dimeric. neither the

KS-A T fragment, nor any of the ~-carbon processing domains were dimers : so that

it was unclear how the dimeric modules were stabilized.

~~ '~

f igure 1.1 6 : X-ray crystal structure of th e KS-AT d ido ma in from DE BS mod ule :; (S mith a nd Tsa i, 2007).

The first X-ray crystal structure from the PKS family was of a 194 kDa

fragme nt from DEBS modul e 5, which encompasses the KS and AT domains

(Figure 1.16) (Tang et aI., 2006). Th is structure came immediately after the

mammalian F AS structu re and showed remarkable similarity with the equiva lent

domains of the F AS protein (Maier et aI. , 2006). The higher reso lution of the

structu re (2.73A) revealed some surprising features in the linker regions flanking

the catalytic domains. A coiled coil preceding the KS domain j uts out of the

structure and is known to play an important recognition role in docking complexes.

The KS-AT linker consists of three ~-strand s and two helices. A 30 residue linker

C-terminus to the AT domain was found to wrap back over the AT domain and the

KS-A T linker to make intimate contacts with the KS domain. The structure

exhibits an extensive dimer interface compris ing of the KS catalytic doma ins and

- 48 -

Chapter 1

the N-terminal coiled-coil structure that protrude outwards away from the catalytic

KS domains. When the structures of the DEBS KS-AT and FAS are overlapped

with the KS domains aligned to each other, the positions of the corresponding AT

domains are almost identical, with a twist of ~10° from the 2-fold axis. This

highlights the structural similarity of PKS with F ASs, the only difference being the

absence of the N-terminal coiled coil docking element in F AS. This implies a

close structural, functional and evolutionary relationship between the two families

of enzymes.

In modular PKSs that lack DB and ER domains, the KR domain is located

adjacent to a core region after the AT domain. This region is also present in all

F ASs and in PKS modules that include both DB and ER domains, but in these

situations the ER domain is inserted between the core and the KR domain.

Attempts to engineer hybrid PKSs by KR replacement were successful only when

part of the core region was included. Also, mutagenesis of the core region in

F ASs, eliminated binding of NADPB to the KR domain located ~400 residues

downstream. These studies suggested that there might be some structural elements

in the core region that are important for KR function. Further insights were

provided by crystallographic studies on an engineered DEBS 1 protein by

Keatingle-Clay and Stroud (Keatinge-Clay and Stroud, 2006). The structure

included both the KR domain and upstream core sequences and revealed two

subdomains, each similar to a short-chain dehydrogenase/reductase (SDR)

monomer. The first domain belonged to the core region and had a truncated

Rossmann fold. It appeared to perform a strictly structural role in stabilizing the

catalytic KR subdomain. This implied that the KR coding region is interrupted by

- 49-

Chapter 1

insertion of an ER domain in modules that contain a full complement of ~-carbon

processing enzymes.

Secondary structure prediction also suggested that the region downstream

of the AT domain in module 4 adopts a double hot dog fold corresponding to the

sequence previously assigned to the DH domain. Thus, it was concluded that the

DH domain was likely a pseudodimer and part of the core region actually

represents the second of the DH pseudosubunits. Thus in a complete PKS module,

the unassigned sequence previously characterized as the core region actually may

represent the second subdomain of the DH domain followed by the structural

subdomain of the KR. The PKS domain organization can thus be defined as KS­

AT-DH-DHpeseudo-KRstructural-ER-KRcatalytic-ACP (Gokhale et aI., 2007a; Keatinge­

Clay and Stroud, 2006).

1.3 FAAL ENZYMES: FUNCTIONAL LINK BETWEEN FASs AND PKSs

F AALs belong to the acyl activating family of enzymes (AAE) and include

the adenylation domains ofNRPSs and fatty acyl-CoA ligases (FACL) (Admiraal

et aI., 2003; Eppelmann et aI., 2002; Trivedi et aI., 2004). While adenylation

domains and FAALs are involved in activation of substrates as adenylates, FACL

perform an additional reaction of conversion of the fatty acyl-adenylate to acyl­

CoA. Interestingly, both the F AAL and F ACL enzymes were earlier cla'lsified into

the same acyl-CoA synthetase family and were referred to as FadD (the D gene of

the fatty acid degradation operon from E. coli) proteins (Black et aI., 1992).

However, biochemical reconstitution of these proteins from Mtb revealed that the

F AAL enzymes are not capable of catalyzing the second reaction of conversion of

the adenylate intermediate to the corresponding CoA (Trivedi et aI., 2004). Even

- 50-

Chapter 1

more remarkable was the fact that many of F AAL homologues were present

adjacent to PKS genes in the Mtb genome (Cole et aI., 1998). Systematic analysis

revealed that the FAAL activated acyl-adenylates are transferred to the PKS

enzymes as starter units and F AALs provide a functional link between the F AS and

the PKS biosynthetic machinery.

Structural analysis of AAEs reveals a conserved fold containing a large N-

terminal and a small C-terminal domain, which undergo domain movements during

various steps of catalysis. A recent report from our lab suggests a mechanism by

which Mtb may have evolved F AAL proteins from the omnipresent F ACLs. The

new catalytic function is said to originate by a substrate-induced conformational

rearrangement in the F ACL proteins. F AAL proteins do not perform the second

reaction of acyl-CoA formation due to the inability of F AALs to generate a

conformation that could utilize CoASH effectively. This is due to the presence of

an insertional motif in the N-terminus of F AAL proteins that modulates the

mobility of the C-terminus and thus abrogates CoA formation (Arora et aI., 2009).

1.4 MYCOBACTERIUM TUBERCULOSIS

Mtb is the etiologic agent of the human disease tuberculosis and remains a

major cause of morbidity and mortality worldwide. Mtb bacilli are encapsulated

by a complex cell envelope known to actively contribute towards virulence. This

waxy barrier also provides protection to the bacterium from various therapeutic

agents (Draper, 1998; Jarlier and Nikaido, 1994). Much of the early work on the

chemical nature of the cell wall of Mtb was carried out by R.J.Anderson and

T.B.Johnson at Yale University, where they focused on characterization of fat-like

material present in the tubercle bacillus. While the initial instinct was for thf­~

- 51 -

Chapter 1

presence of sterols, examination of both aqueous and solvent fractions did not yield

any sterol containing compounds. Purification and careful analysis of these

fractions revealed many new and interesting chemical substances not usually found

in the plant and animal kingdoms. These studies provided early leads for the

presence of trehalose-containing compounds, phthiocerols, mycolic acids, etc in

mycobacteria (Anderson, 1941; Goren, 1972; Minnikin, 1982). Complete

chemical characterization of these complex metabolites was carried out in

subsequent years with the advent of new technologies and methodologies. It is

now established that these chemical compounds are embedded as a highly complex

network of sugars, proteins and lipids in the mycobacterial cell wall (Daffe and

Draper, 1998; Lederer et ai., 1975).

1.4.1 The mycobacterial cell wall

The base of the mycobacterial cell wall consists of a plasma membrane,

which can be resolved into a thick outer layer and a thin inner layer using electron

microscopy (Figure 1.17) (Paul and Beveridge, 1992; Zuber et ai., 2008). The

thickness of the outer layer is associated with the presence of carbohydrates and

phospholipids, including phosphatidylinositol mannosides (PIMs). PIMs are

involved in anchoring polysaccharides like lipoarabinomannan (LAM) and

lipomannan (LM) in the cell wall (Brennan, 1988; Daffe and Lemassu, 2000;

Guerardel et ai., 2002; Pitarque et ai., 2008). The plasma membrane also hosts a

variety of other compounds like carotenoids, menaquinones and various

glycosylphosphopolyprenols (Brennan and Nikaido, 1995; Daffe and Lemassu,

2000). Outside the plasma membrane, a complex polymer of peptidoglycan

surrounds the mycobacteria and acts as a scaffold to which arabinogalactan

- 52-

Chapter 1

moieties are connected by L-rhamnose-D-N-acetylglucosamine. The C-l of N-

acetylglucosamine is phosphodiesterified to the hydroxyl moiety of C-6 of the

muramic units of the peptidoglycan layer. The rhamnose moiety is connected to

the galactan of the arabinogalactan layer, generating a bridge between the

arabinogalactan and the peptidoglycan layer. Mycolic acids are esterified to these

distal arabinose sugars of the arabinogalactan layer (Brennan, 2003; Brennan and

Nikaido, 1995; McNeil et at, 1991; Misaki et at, 1974). Mycolic acids are also

present as free mycolates or as esters of trehalose sugars called trehalose mono-

mycolates (TMM) and trehalose dimycolates (TDM) (Bloch and Noll, 1955; Noll

et at, 1956; Takayama et at, 2005). A number of other surface-exposed lipids like

sulfolipids (SL), polyacyltrehaloses (PAT), phthiocerol dimycocerosates (PDIM),

mannosyl-~-l-phosphomycoketides (MPM) and diacyltrehaloses (DAT) intercalate

into the cell wall. This lipid rich cell wall also harbors a number of proteins.,

including the antigen 85 complex and porins, which constitute the complex cell

wall assembly (Asselineau and Laneelle, 1998; Brennan and Nikaido, 1995;

Draper, 1998; Gokhale et at, 2007b).

Porin MPM ,

lM portion of lAM

Galactan ~ ____ ,_ .,.. __ ....... ~_ ... Arabinose sugar

Figure 1.17: Schematic representation of the mycobacterial cell envelope (Chopra and Gokhale, 2009).

- 53 -

Chapter 1

1.4.2 Mycobacterial PKSs and their role in biosynthesis of complex lipids

Initial analysis of wax components from Mtb revealed a mixture of fatty

acids ranging from saturated palmitic acid to a wide variety of methyl branched

fatty acids. These included tuberculostearic acid (10-methyl CI8 fatty acid),

dextrorotatory fatty acids analogous to phthienoic acid (tri-methyl unsaturated C27

acid) and levorotatory fatty acids called mycocerosic acids (tetra-methyl branched

C28 to C32 acids) (Anderson, 1941; Asselineau, 1966; Asselineau and Laneelle,

1998; Brennan, 1988; Goren, 1972). While the structure and chemical nature of

these compounds was elucidated using conventional chemical characterization

techniques, the enzymology and biochemistry of their in vivo biosynthesis was not

well understood.

Key insights into the biosynthesis of branched chain fatty acids were

provided by precursor feeding experiments in Mtb (Gastambide Odier et ai., 1963;

Narumi et ai., 1973; Yano and Kusunose, 1966). Radioactive 14C-propionate/14C­

acetate units are converted to methylmalonyl CoA Imalonyl CoA, respectively, by

biotin-dependent propionyl/acetyl CoA carboxy lases (Rainwater and Kolattukudy,

1982; Rawlings, 1997; Savvi et ai., 2008). These methylmalonate and malonate

units are utilized by the fatty acid synthase (F AS) and PKS enzymatic machineries

for biosynthesis of various metabolites. Lipid analysis of Mtb grown in the

presence of 14C-propionate as a tracer revealed incorporation of radioactive label

into the branched end of mycocerosic acids (Gastambide Odier et ai., 1963).

However, the fatty acid synthase responsible for the biosynthesis of these

methylated acids could not be identified and it took another 20 years before

Kolattukudy and coworkers demonstrated the existence of another elongation

- 54-

Chapter 1

system. This elongation system, referred to as mycocerosic acid synthase (MAS),

resembled the F ASs in all respects, except for exhibiting specificity for

methylmalonyl CoA (Gastambide Odier et aI., 1963; Rainwater and Kolattukudy,

1983). Cloning and expression of the mas gene and construction of a genetic

knockout strain of Mycobacterium bovis BCG facilitated investigation of the in

vivo role of MAS in mycobacterial biology (Azad et aI., 1996; Mathur and

Kolattukudy, 1992). 14C-propionate feeding experiments with this knockout strain

established a role of MAS in biosynthesis of mycosides (phenolic glycolipids), and

this methodology provided a basis for analyzing the in vivo role of mycobacterial

genes in the biosynthesis of various cell wall lipids (Azad et aI., 1996). Moreover,

the presence of other methylated fatty acids in mycobacteria suggested the

existence of multiple mas-like genes (Azad et aI., 1996; Kolattukudy et aI., 1997;

Mathur and Kolattukudy, 1992).

In agreement with the presence of a number of lipid metabolites unique to

Mtb, genome sequencing revealed many genes involved in lipid metabolism (Cole

et aI., 1998; Natarajan et aI., 2008). Apart from the type I and type II F AS systems

involved in fatty acid biosynthesis, several gene clusters homologous to PKSs were

identified. Sequence homology studies suggested mycobacteria to contain

examples of all three polyketide biosynthetic systems. (Cole et aI., 1998; Natarajan

et aI., 2008). Since PKSs from Streptomyces are involved in the biosynthesis of

polyketide natural products (Hopwood, 1997; Katz and Donadio, 1993; O'Hagan,

1992; Sanchez et aI., 2008), the existence of these homologues indicated the

presence of polyketide metabolites in Mtb. Prior to the genome availability,

sequencing around the mas gene cluster had indicated the presence of modular

- 55 -

Chapter 1

PKSs (Azad et aI., 1997; Kolattukudy et ai., 1997). Research over the last decade

has given some fascinating insights into the role of these enzymes in mycobacterial

biology, where they have been implicated in the biosynthesis of various virulence

lipids (Chhabra and Gokhale, 2009; Gokhale et ai., 2007a; Gokhale et ai., 2007b;

Jackson et ai., 2007).

1.4.2.1 PKS12 is involved in the biosynthesis of mannosyl-p-l-phosphomycoketides

Pks12 is the largest open reading frame in the Mtb genome and encodes

two modules that can catalyze the synthesis of a saturated acid. Such large genes,

encoding multiple sets of modules have been characterized only in antibiotic

producing organisms, where they form a part of a large modular PKS cluster. The

functional role of PKS12 in Mtb was probed by generation of a pks12-disrupted

mutant strain and its biochemical analysis using radioactive propionate as a tracer.

These studies suggested that pks 12 could be involved in the biosynthesis of

phthiocerol dimycocerosates (PDIMs) (Sirakova et ai., 2003). However,

subsequent cell free reconstitution studies followed by a careful analysis of the

pks12 mutant strain by another group disproved this hypothesis (Matsunaga et ai.,

2004). It was shown that PKS12 is involved in the biosynthesis of novel antigenic

phospholipids called MPMs. These compounds are present in pathogenic species

of mycobacteria and are chemically similar to the mammalian mannosyl-~-I-

phosphodolichols. They possess an identical mannose-phosphate head group but

differ in their alkyl chain, which probably contributes to antigenicity of MPMs

(Moody, 2001). While initial studies had suggested an isoprenoid mode of

biosynthesis for the alkyl segment (now referred to as the mycoketide), careful

- 56-

Chapter 1

mass spectrometric analysis ofMPMs and gene inactivation revealed PKS12 to be

involved in the biosynthesis of these compounds (Matsunaga et aI., 2004; Moody

et aI., 2000). The two modules of PKS12 were predicted to exhibit

methylmalonate and malonate extender unit specificity and thus mycoketide

biosynthesis was proposed to involve five alternate condensations of

methylmalonyl and malonyl units by using an iterative mechanism of biosynthesis

(Matsunaga et aI., 2004). The mycoketide chain undergoes offloading from the

protein, is reduced, phosphorylated and glycosylated by an unknown mechanism to

yield the final phospholipid antigen (Figure 1.18).

PKS12 r\.

PKS12

--------t _1 ~.. 4- l.r.:, ..t {001~

----

HO~R MycoI<etide alcohol

!? R

Mannosyl~ 1-pOOsptto mycoketide (MPM)

5 o

Figure 1.18: The proposed MPM biosynthetic pathway (Chopra and Gokhale, 2009).

The suggested iterative mechanism of catalysis for the formation of the

mycoketide chain would require transfer of the growing chain from the ACP of the

second module to the KS active site of the first module. This implies a covalent

- 57-

Chapter 1

transfer of acyl chains over very large distances and based on the three-

dimensional organization of F ASs and PKSs, seems unlikely (Chopra et aI., 2008;

Khosla et aI., 2007; Maier et aI., 2006; Sherman and Smith, 2006). Thus, the

biosynthesis of MPMs by PKS12 presents an interesting challenge to the

intramolecular paradigm of iterative catalysis. In this thesis, we have attempted to

understand the process of iterative condensations in the biosynthesis of MPMs and

have discovered a novel "modularly-iterative" mechanism of polyketide

biosynthesis.

1.4.2.2 PKS1511, PpsABCDE & MAS are involved in biosynthesis of dimycocerosate esters

Analysis of lipid extracts suggested a number of hydroxy compounds in

mycobacteria (Asselineau, 1966). Amongst these, the methoxyglycols were

termed as phthiocerols (3-methoxy, 4-methyl 9, II-dihydroxy glycols) and were

found to be esterified with mycocerosic acids. Interestingly, bovine strains of

mycobacteria were found to contain a glycolipid called mycoside B, which on

hydrolysis yielded mycocerosic acids and a variant of phthiocerol called

phenolphthiocerol (p-glycosylated phenylglycol). Phenolphthiocerol possesses a

phthiocerol like chain but ends with a phenolic moiety glycosylated with various

sugars (Asselineau, 1966; Onwueme et aI., 2005a). Together, the phthiocero)

esters and the phenolphthiocerol esters are referred to as DIMs. These esters are

present on the cell surface of Mtb and have been implicated in its virulence

(Camacho et aI., 1999; Cox et aI., 1999; Reed et aI., 2004). Knockout studies

provided early insights into the biosynthesis of these compounds. While the mas-

disrupted mutant of M. bovis BCG was incapable of synthesizing mycocerosic

- 58-

Chapter 1

acids (Azad et ai., 1996), the pps-disrupted mutant lacked PDIMs and phenolic

glycolipids (PGLs) (Azad et ai., 1997). Genome sequencing revealed that the

genes involved in biosynthesis of these metabolites are clustered in a large 73-kb

operon (Cole et ai., 1998; Onwueme et ai., 2005a). A combination of genetic and

biochemical studies have now provided a comprehensive picture for biosynthesis

of these compounds (Figure' 1.19). The biosynthesis of PGLs and PDIMs can be

dissected into four steps: 1. priming of PpsA with appropriate PGL- or PDIM·

specific starter unit; 2. extension of the primer unit by PpsABCDE, leading to the

generation of the diol; 3. biosynthesis of mycocerosic acids by MAS; 4.

esterification and final assembly.

The biosynthesis of PGLs is initiated by utilizing a common metabolic

intermediate, chorismate, which is converted to p-hydroxybenzoic acid (PHB) by a

chorismate pyruvate-lyase, Rv2949c (Stadthagen et ai., 2005). pHB is then

activated and transferred to a type I iterative PKS15/1 enzyme by FAAL22

(Ferreras et ai., 2008). PKS1511 exhibits extender unit specificity for malonyl CoA

and is believed to extend pHB to p-hydroxyphenylalkanoate (Figure 1. 19a). The

H37Rv strain of Mtb is devoid of PGLs due to a frameshift mutation in this gene

(Constant et at, 2002; Reed et ai., 2004). The Beijing family of Mtb strains, as

well as M. bovis BCG and Mycobacterium leprae, possess a functional copy of this

gene and make PGLs (Daffe and Laneelle, 1988; Reed et ai., 2004; Tsenova et at.,

2005). The p-hydroxyphenylalkanoate chain is then transferred to the PpsA starter

for PGL biosynthesis. The starter n-fatty acyl units for PDIM synthesis are

provided by FAAL26, which also uses the novel acyl-adenylate activation

- 59-

Chapter 1

mechanism (Trivedi et aI., 2004). From this stage onwards, the biosynthesis of

PGLs and PDIMs follows the same biosynthetic route (Figure 1.19b).

(a)

Figure 1.19: (a) Biosynthesis of pHB and loading onto PKS 15/1. (b) Biosynthesis of the diol component of PDIMs and PGLs by PpsABCDE. (c) Biosynthesis of mycocerosic acids by iterative MAS and condensation with diol for the formation of PGLs and PDlMs. The ER domain in PpsD is a trans ER, which is not a part of the type I architecture ofPpsD. For simplicity, it has been shown within the PpsD domain organization (Chopra and Gokhale, 2009).

PpsA catalyzes extension of the starter units with malonyl CoA, which

results in the formation of a mono-hydroxy fatty acid. This is due to the presence

of a single KR auxiliary domain in PpsA. The acyl chain thus generated is then

transferred to PpsB, which catalyzes formation of a diol through another 2-carbon

condensation, followed by ketoreduction. PpsC adds a malonyl unit to the growing

chain and also catalyzes complete reduction to a methylene group. PpsD, in

conjunction with a trans-acting ER, Rv2953, extends this chain further with a

methylmalonyl moiety (Simeone et aI., 2007; Trivedi et aI., 2005). The final

- 60-

Chapter 1

extension to a phenolphthiocerol or phthiocerol chain is performed by PpsE, which

can utilize either malonyl CoA or methyl malonyl CoA extender units (Trivedi et

al., 2005). MAS protein possesses all three auxiliary domains (KR, DH and ER)

necessary for complete reduction of newly-generated ~-carbonyl acyl chain. MAS

carries out iterative condensation of multi-branched fatty acids by using medium­

to long-chain fatty acyl-CoA starters with methylmalonyl CoA extender units

(Mathur and Kolattukudy, 1992; Onwueme et al., 2004; Trivedi et al., 2005).

Polyketide-associated protein AS (PapAS) interacts with MAS and brings about

trans-esterification of mycocerosic acids onto the diol component of

phthiocerol!phenolphthiocerol (Figure 1.19c) (Mathur and Kolattukudy, 1992;

Onwueme et al., 2004; Trivedi et al., 2005).

Final processing and transport of DIMs requires other proteins like

Rv29S1c and Mtf2, which bring about reduction of the keto-group and subsequent

O-methylation of this hydroxyl group (Onwueme et al., 200Sb). The glycosylation

of the phenyl ring in PGLs is carried out by glycosyl transferases, Rv2962c,

Rv29S8c and Rv29S7c, which modify the phenyl ring with addition of two

rhamnose and one fucose sugars (Perez et al., 2004b). The fucose ring is further

modified by action of methyl transferases, which complete the assembly of tri-O­

methyl di-rhamnosyl-phenolphthiocerol dimycocerosates (Perez et al., 2004a).

The transport of the fully assembled DIMs to the cell wall is proposed to be

mediated by a transmembrane protein, MmpL7, which is thought to couple

synthesis with transport by specifically interacting with PpsE (Jain and Cox, 2005).

DrrC and LppX are other accessory proteins that mediate the transport of DIMs to

the periphery of the cell wall (Onwueme et al., 200Sa; Sulzenbacher et al., 2006).

- 61 -

C/wpter 1

1.4.2.3 PKS2 is involved in the biosynthesis of sulfolipids

Sulfolipids (SLs) were identified in the late 1950s from Mtb while studying

a sulfur-containing material capable of fixing the cationic dye neutral red (Dubos

and Middlebrook, 1948; Middlebrook et aI., 1959). Subsequent analysis of this

material by Goren and coworkers revealed a mixture of highly related compounds,

with the most abundant being sulfolipid-I (SL-I). Chemical analysis revealed a

trehalose-2-sulfate (T2S) core, tetra-acylated with fatty acids. While one of the

fatty acid substituent is a straight-chain fatty acid (primarily palmitate or stearate),

the other three are long-chain methylated fatty acids called phthioceronic acid (PA)

or hydroxy phthioceronic acids (HP A) (Goren, 1970a; Goren, 1970b; Goren et aI.,

1976; Goren et aI., 1971). Phthioceronic acids differ from mycocerosic acids in

having an absolute configuration of S- (also referred to as L- based on the older

nomenclature) for the methyl branched carbon, as compared to R- (or D-) in the

case of mycocerosic acids (Asselineau, 1966). Gene inactivation and

complementation studies clearly indicated an essential role for pks2 in the

biosynthesis of these unusual acids (Sirakova et aI., 2001). PKS2 possesses a

complete set of active sites to add a completely reduced ketide unit to the starter

chain. In this thesis, we demonstrate that PKS2 is indeed involved in the

biosynthesis of long-chain branched fatty acids by iterative utilization of

methylmalonyl CoA. Genetic inactivation studies demonstrate that FAAL23 is

necessary for the biosynthesis of SLs (Lynett and Stokes, 2007) and studies from

out laboratory show that this protein is involved in activation and loading of starter

fatty acyl substrates on to PKS2.

- 62-

Chapter 1

The biosynthetic steps of sulfolipid biosynthesis have been deciphered by

generating mutants of Mtb lacking various enzymes involved in SL biosynthesis

(Figure 1.20). The biosynthesis is initiated by the sulfotransferase StfO, which

transfers a sulfuryl group from 3' -phosphoadenosine-5' -phosphosulfate (Po APS)

onto trehalose, thereby generating trehalose-2-sulfate (T2S) (Mougous et aI.,

2004). T2S is acy lated either by a palmitate or a stearate unit at the 2' -position by

PapA2 to produce mono-acylated SL. This is followed by PapAl-mediated

transfer of the phthioceranoyl group from PKS2 to the palmitoyl-/steawyl-T2S,

leading to the formation of the diacylated intermediate (Bhatt et aI., 2007b; Kumar

et aI., 2007). Since PKS2 lacks an appended TE domain for chain rdease, one

would expect PapAl to interact with and sequester the chain from PKS2 for its

transesterification onto mono-acylated sulfolipids.

?

R, c ·(eH,),..-cI\

OIACYtATEO 51.

51.1

Figure 1.20: Assembly of SL-l. Phthioceronic acids are biosynthesized by an iterative PKS2 protein and are utilized for SL-l production (Chopra and Gokhale, 2009).

- 63 -

•

Chapter 1

A transmembrane protein called MmpL8 is also present in the SL cluster

and is believed to play an important role in transport of SLs across the cell wall.

Disruption of MmpL8 by two independent groups led to the accumulation of

diacylated intermediates, SL1278 or SL-N (Converse et aI., 2003; Domenech et aI.,

2004). The conversion of di-acylated sulfolipid to tetra-acylated mature sulfol'ipids

requires further investigation. The exact biological function of SL-I in

mycobacterial biology is not clear and Mtb mutants of various enzymes involved

in sulfolipid biosynthesis have not provided clear correlation to its virulence

(Bertozzi and Schelle, 2008). Interestingly, a sulfolipid deficient PKS2 knockout

strain of Mtb retains its ability to stain with neutral red (Andreu et aI., 2004;

Cardona et aI., 2006).

1.4.2.4 PKS13 catalyzes condensation of fatty acyl chains during biosynthesL~ of mycolic acids

Mycolic acids are the most abundant lipids found in the mycobacterial cell

wall and are responsible for the "acid fast" nature of mycobacteria. Initial

characterization by Anderson in the late 1930s suggested a general formula of

C88H 1760 4, and a characteristic property to yield normal hexacosanoic acid on

pyrolytic distillation under vacuum (Asselineau, 1966; Asselineau and Laneellle,

1998). Detailed chemical characterization over the years has revealed them to be

a-alkyl-~-hydroxy fatty acids, which are present either as mycolyl-esters or as free

fatty acids in the cell wall (Asselineau and Lederer, 1950; Brennan and Nikaid0,

1995; Goren, 1972). The mycolate structure can be broken down to a saturated

alkyl chain condensed to a longer meromycolate chain, which may carry various

modifications. These modifications on the meromycolate chain classify mycolic

- 64-

Chapter]

acids into alpha-, keto-, and methoxy- subgroups. a-Mycolic acids contain two

cyclo-propane rings on the meromycolate chain and are the major type of mycolic

acids in most mycobacterial species. Keto- and methoxy- mycolic acids carry

additional oxygen functionalities in the meromycolate chain and are often termed

oxygenated mycolates (Barry et ai., 1998; Minnikin and Polgar, 1967; Takayama

et ai., 2005; Toubiana et ai., 1979). Mycolic acids have also been found to contain

unsaturation in the meromycolate chain. Thin layer chromatography and mass

spectrometric approaches for mycolic acid characterization now provide a means

to discriminate between many closely related mycobacterial species (Asselineau

and Laneelle, 1998; Marrakchi et ai., 2008; Minnikin et ai., 1984; Takayama et ai.,

2005).

The biosynthetic pathway for mycolic acids utilizes both the type I F AS

and the type II F AS machinery for the synthesis of the chains; and a PKS called

PKS 13 for their condensation (Figure 1.21). The biosynthesis can be described in

three steps: 1. Type I F AS-mediated biosynthesis of both the alpha-alkyl chain and

the meromycolate precursor; 2. Extension and modification of the meromycolate

chain by the type II FAS biosynthetic machinery; 3. Condensation of the two

chains and export to the cell wall.

Rv2524c is the type I F AS that utilizes acetyl CoA as the starter and carries

out repetitive decarboxylative condensations with malonyl CoA to biosynthesiZle

the hexacosanoyl CoA (alpha chain) and the meromycolate precursor (Blodl,

1977; Smith et ai., 2003). The ketosynthase, FabH (Rv0533c), links the type I and

II F AS pathways and catalyzes condensation of type I F AS-derived meromycolate

precursor with malonyl units present on AcpM (Choi et ai., 2000a; Schaeffer et a},.,

- 65 -

Chapter 1

2001a). The generation of malonyl-AcpM is catalyzed by the enzyme FabD

(Kremer et aI., 2001). Condensation of meromycolate precursor with malonyl

CoA leads to chain extension by two units and is subjected to a cycle of keto··

reduction, dehydration and enoyl-reduction, catalyzed by MabA (FabG1, Rv1483)

(Marrakchi et aI., 2002a), AcpM dehydratase (Cronan et aI., 1988; Gurvitz et aI.,

2008b; Sacco et aI., 2007), and InhA (Rv1484) (Gurvitz et aI., 2008a), respectively

(Figure 1.21). InhA is inhibited by the antituberculosis drug isoniazid via

formation of a covalent adduct with NAD+ (Cronan et aI., 1988; Dessen et aI.,

1995; Gurvitz et aI., 2008a; Gurvitz et aI., 2008b; Marrakchi et aI., 2002a;

Marrakchi et aI., 2000). The extended chain is transferred to KasA (Rv2245) and

KasB (Rv2246), which catalyze further extension using the same sets of enzymes

(Bhatt et aI., 2005; Bhatt et aI., 2007a; Kremer et aI., 2002; Schaeffer et aI., 2001b;

Slayden and Barry, 2002). It is proposed that while FabH catalyzes the initial

condensation, KasA carries out extension to an intermediate stage, followed by

extension to full length meromycolate by KasB. The modifications in the

meromycolate chain are brought about by various cyclopropane synthases and

methyl transferases during the type II F AS-mediated chain extension cycles (Bhatt

et aI., 2007a; Marrakchi et aI., 2008; Takayama et aI., 2005).

The condensation of the meromycolate and the alpha chain is brought about

by PKS13, which has domain architecture ofACP-KS-AT-ACP-TE (Gokhale et

aI., 2007b; Portevin et aI., 2004). The meromycolate chain from meromycolyl­

AcpM is activated by FAAL32 and transferred to the KS domain of PKS13

through the N-terminal ACP domain (Trivedi et aI., 2004). It is not clear if

F AAL32 directly interacts with AcpM and sequesters the chain or the chain is

- 66-

Chapter 1

released prior to activation by F AAL32. Hexacosanoyl CoA, derived from the

type I FAS pathway, is acted upon by two acyl-CoA carboxylases, AccD4 and

AccD5, leading to formation of 2-carboxy-hexacosanoyl CoA (Gande et aI., 2007;

Gande et aI., 2004). Through the AT domain of PKS13, the 2-carboxy-

hexacosanoyl CoA is transferred to the ACP domain where it undergoes a

decarboxylative Claisen condensation with the meromycolate chain. Rv2509 is

believed to carry out the final reduction of the p-keto group to a secondary alcohol

for the formation of mature mycolates (Figure 1.21) (Bhatt et aI., 2008; Lea-Smith

et aI., 2007).

Figure 1.21: Biosynthesis of mycolic acids requires formation of meromycolate-chain and a­chain through the type I FAS/type II FAS systems, followed by condensation by PKS13 (Chopra and Gokhale, 2009).

- 67-

Chapter 1

It is proposed that mycolic acids are transferred from the ACP domain of

PKS 13 to mannopyranosy 1-1-phosphoheptaprenol (PL), which transfers the

mycolyl-group further to trehalose-6-phosphate to yield trehalose-mono-mycolyl

(TMM) phosphate. Dephosphorylation of TMM-phosphate leads to formation of

TMM, which is exported out to the cell wall. Enzymes responsible for transfer of

mycolates from PKS13 to the cell wall have not been very well characterized. The

extracellular mycolyl transferases called the Ag85 complex are proposed to

catalyze the formation of TDM and arabinogalactan-mycolate from TMM (Bhatt et

aI., 2007a; Marrakchi et aI., 2008; Takayama et aI., 2005). Recent studies suggest

new possible modes of lipid biosynthesis involving formation of long-chain fatty

acids including mycobacteric acids by degradation of mycolic acids (Rafidinarivo

et aI., 2008).

1.4.2.5 PKS314 is involved in the biosynthesis ofphthenoic acids

One of the components of the Mtb wax fraction analyzed by Anderson and

coworkers were dextrorotatory fatty acids called "phthioic acids" esterified to a

sugar. These acids were initially thought to be completely saturated tri-methylated

fatty acids. Detailed chemical characterization by two independent research

groups revealed their chemical nature as tri-methylated a,~-unsaturated acids or 2,

4, 6-tri-methyltetra-cos-2-enoic acids. These acids were independently referred to

as phthienoic acids or . mycolipenic acids by the two groups (Figure 1.22)

(Asselineau et aI., 1972; Asselineau, 1966).

During the late 1980s, analysis of mycobacterial glycolipids revealed a

penta-acylated compound where four phthienoyl-groups and one palmiit.oyl- or

stearoyl- group were found to occupy the 2,2',3',4, and 6' positions of trehalose

- 68-

Chapter 1

sugars (Daffe et al., 1988). These compounds were termed polyacyltrehaloses

(PATs) and were found in virulent human and bovine strains of mycobacteria

(Figure 1.22). Similarly, a number oftri-acylated trehalose (TATs) and diacylated

trehalose (DATs) compounds containing various different acylations were

identified (Besra et al., 1992; Cruaud et al., 1990; Lemassu et al., 1991; Munoz et

al., 1997a; Munoz et al., 1997b).

o

C17H35

Figure 1.22: Structure of polyacyltrehaloses. One of the phthienoic acids is highlighted in the box (Chopra and Gokhale, 2009).

Biochemical analysis of a PKS3/4 mutant strain of Mtb suggested

involvement of PKS3/4 in the biosynthesis of phthienoic acids. Interestingly,

H37Rv genome sequencing had suggested pks3 and pks4 to be independent open

reading frames. Subsequent analysis identified an error in sequencing and showed

PKS3/4 to be a single protein with KS-AT-DH-ER-KR-ACP domain organization

(Dubey et a1., 2002). The absence of PATs from the Mtb strain caused cells to

stick to each other as a clump without affecting the overall growth rate (Dubey et

a1., 2002). This suggested PATs to be localized on the outer surface of the cell

wall. In another study, a pks3/4 mutant Mtb strain showed improved efficiency of

binding to the host cells (Rousseau et al., 2003a). However, this property did not

affect the overall replication and persistence of the bacillus in the host cells.

- 69-

Chapter 1

The biochemical pathway involved in assembly of PATs has not been

investigated. Analysis of the pks3 and pks4 genetic locus reveals genes which

could participate in the complete assembly and transport of these lipids. F AAL2l

could activate and load fatty acyl chains onto PKS3/4 for the biosynthesis of

phthienoic acids. Also encoded in the cluster is a PKS-associated protein, PapA3,

which could mediate trans-esterification of phthienoic acids onto trehalose sugars

and MmpLl 0 for export of PATs to the outer cell wall.

1.4.2.6 PKSI0, PKS7, PKS8, PKS17, PKS9 and PKSll constitute an unusual pks cluster

The H37Rv genome encodes three genes homologous to type III PKSs.

Interestingly, two of these genes, pks 10 and pks 11, are present on either side of

four type I PKSs, constituting a pks cluster (pksIO-pks7-pks8-pksI7-pks9-pks11)

(Figure 1.23) (Cole et aI., 1998). PKS7 is ~3l % identical to MAS and contains all

the three auxiliary domains that could completely reduce a ketide unit. PKS8

contains KS, AT, DH and ER domains and PKS17 contains KR and ACP domains.

Together, PKS8 and PKS17 would form one complete module. PKS9 resembles

loading modules of modular PKSs and comprises KS, AT and ACP domains. In

PKS9, the active site cysteine ofKS is mutated to glutamine and is a KSQ domain.

The genome of M. bovis has also revealed an identical genomic organization of the

pkslO-pks11 cluster, with the putative proteins sharing 98-100% sequence identity

with the Mtb homologues (Gamier et aI., 2003). However, M. avium subsp.

paratuberculosis shows slight variations in the genomic organization and the

broken module is missing from the cluster (Figure 1.23) (Li et aI., 2005).

- 70-

Chapter 1

PKSfD PKS7 PKS8 PKSf7 PKS9 PKS11

M. tuberculosis -~~~~~ ~~~-~,~ ~"-- --------~- --~ --- ~ .. ,. ~ 1 n. '. ,L ." ... t ~:.f ~ to! ~'" If '!B;. i':lT-t: I ~

PKS10 PKS7 PKSB PKS17 PKS9 PKS11

M. bovis --;- ~~~.-~~- .. ~~-- ----_. -- ~

M. ' ~-..: l ~;' ~ ~ . -., ~ t ~ u .~ ••• ...~. ~~ _11 /~... "~~1

PKS1D PKS7 PKS11

M. avium ssp. paratuberculosis

~--{.-- --~~~~.--.-- --- ~ "', ~ Ten~. 4' ¥.. ,. '\JI'" TiIo .. ;. l j

Figure 1.23: Organization of the PKSIO-PKSll cluster in various mycobacterial species (Chopra and Gokhale, 2009).

The functional importance of the pksIO-pksll genomic cluster is yet to be

established in Mtb and M. avium subsp. paratuberculosis. Gene inactivation

studies suggest a possible role of pks7 and pks11 in the biosynthesis of PDIMs

(Rousseau et ai., 2003b; Waddell et ai., 2005). This could also be due to

spontaneous loss of PDIMs from the mutant strains (Domenech et ai., 2004).

Another report suggested a role for pks8 and pks17 in the biosynthesis of methyl

branched unsaturated fatty acids that are esterified to acyltrehaloses and sulfated

acyltrehaloses as minor constituents (Dubey et ai., 2003). Biochemical

characterization of PKSll suggests that it may be able to produce resorcinolic

metabolites, which are known for their involvement in cellular physiology and

membrane chemistry in other organisms (Saxena & Gokhale, unpublished results)

(Kozubek and Tyman, 1999). These amphiphilic molecules possess diverse

biological functions and are active antimicrobial and antiparasitic compounds.

They are also known to modulate oxidation of liposomal membranes and fatty

acids (Gubemator et aI., 1999). Interestingly, early analysis of unsaponifiable

fraction of fats from M. leprae has demonstrated the presence of methoxylated

resorcinolic metabolites called (1- and ~-leprosols (Asselineau, 1966; Bu'Lock and

Hudson, 1969). Similar molecules have been recently shown to be essential for

- 71 -

Chapter 1

fonnation of metabolically dormant cysts in Azotobacter vinelandii (Funa et ai.,

2006). It is tempting to speculate that these metabolites may be produced under

specific conditions in mycobacteria and may have a role to play in the onset of

donnancy.

1.4.2.7 PKS18 is involved in the biosynthesis of long-chain pyrones

The third type III pks gene, pks18, is not flanked by PKS-related genes and

shows 40-45% sequence homology with bacterial and plant type III PKSs.

Sequence analysis of PKS18 shows conservation of the catalytic and key active

site residues of this class of proteins (Cole et ai., 1998). While no physiological

role has been assigned to pks 18, biochemical investigation of its product revealed

remarkable specificity for long-chain aliphatic CoA analogues. This unusual

substrate specificity is unprecedented in the chalcone synthase super-family of type

III PKSs and has added a new functional relevance to these proteins. PKS 18

efficiently produces long-chain a-pyrones when primed with the long fatty acyl-

CoAs (Rukmini et ai., 2004; Sankaranarayanan et ai., 2004; Saxena et ai., 2003).

Since the biochemical characterization of PKS 18, a number of plant and bacterial

homologues have been shown to utilize long-chain precursor molecules for the

synthesis of acyl pyrones. Such acyl pyrones have been recently identified in the

cell envelope of Azotobacter (Abe et ai., 2005; Abe et ai., 2004; Austin et ai.,

2004; Funa et ai., 2006; Zha et ai., 2006).

1.4.2.8 MbtC and MbtD are involved in the biosynthesis of iron-chelating siderophores

Siderophores are iron-chelating compounds (see the chapter by Kadi and

Challis in this series) that were discovered in mycobacteria in the late 1950s while

- 72-

Chapter 1

searching for factors that are important for the growth of M. paratuberculosis

(Snow, 1970). Subsequent research has revealed two types of siderophores which

collectively scavenge iron in the Fe (III) form from the host organism.

Mycobactins or intracellular siderophores are found within the cell envelope of the

mycobacteria and are believed to playa role in controlled release of Jiron inside the

cell. Extracellular siderophores vary in composition and are called

carboxymycobactin or exochelins depending upon whether the organism is

pathogenic or saprophytic (Ratledge, 2004; Ratledge and Marshall, 1972;

Rodriguez, 2006).

Chemically, mycobactin and carboxymycobactin have a central lysine core

which is modified at both the (1- and £-amino termini with a hydroxyaryloxazoline

group and an alkyl group, respectively (Figure 1.24). The alkyl group varies from

CIO to C2l in the case of mycobactin and sometimes contains a cis-double bond.

However, it is shorter in the case of carboxymycobactin and carries a free carboxy

group at the end. It is this alkyl group which differentiates carboxymycobactin

from mycobactin. On the carboxyl-end, lysine is modified with a polyketide-

\

derived ~-hydroxy butyrate group, which is further linked to another N-

hydroxylated and cyclized lysine. All three modifications on lysine together

constitute the iron-coordinating framework of mycobactins (Ratledge, 2004).

Although the biochemical reconstitution of mycobactin assembly has not

been carried out, two gene clusters: mbtl and mbt2 are proposed to be involved in

its biosynthesis (Quadri et aI., 1998). Expression of these two clusters is believed

to be regulated by an iron-dependent IdeR repressor (Rodriguez, 2006; Rodriguez

and Smith, 2003). mbtl codes for MbtA to MbtJ, believed to participate in the

-73 -

Chapter 1

assembly of the polyketide-peptide core (Quadri et aI., 1998). mbt2 codes for

enzymes responsible for modification of this core to the final metabolite (Krithika

et aI., 2006). The predicted pathway for assembly of mycobactin starts with the

conversion of iso-chorismic acid to salicylic acid, which is activated by MbtA, a

salicylate-AMP ligase, and loaded onto MbtB. MbtB is a non-ribosomal peptide

synthetase and is expected to attach a Ser or Thr onto the salicylic acid core. It is

this amino acid which is cyclized to an oxazoline ring, thus finishing the covalent

assembly of the a-amino cap of lysine. MbtE is another NRPS and is thought to

catalyze the addition of the core lysine onto this cap (De Voss et aI., 1999;

Marshall and Ratledge, 1972; Snow, 1970).

mbt-2 mbt-1

~ ~~~~~~--~~

MycobacI:'" n_17. 19; R-CH.

Carboxymycobac1in: n=2·9; RcCOOH

Figure 1.24: The mbt locus involved in the biosynthesis of mycobacteria. The structures of mycobactinlcarboxymycobactin are shown. The core lysine is shown in bold (Chopra and Gokhaie, 2009).

MbtC and MbtD are the two polyketide synthase subunits present in the

cluster and code for the KS and AT-KR-ACP domains respectively (Figure 1.24).

Together, they constitute a PKS enzyme believed to carry out the biosynthesis of

the P-hydroxy butyrate group using acetyl and malonyl CoA units. Another NRPS

called MbtF is proposed to transfer the final lysine onto the p-hydroxy butyrate

group. The cyclization of this N-hydroxylated lysine group to a seven-membered

- 74-

Chapter 1

lactam ring is proposed to be catalyzed by MbtJ (De Voss et aI., 1999; Quadri et

aI., 1998). Modification of the €-amino termini of the core lysine is brought about

by the mbt2 cluster, which codes for an N-acyl transferase (MbtK), an acyl carrier

protein (MbtL), a fatty acyl-AMP ligase (FAAL33 or MbtM) and an acyl-CoA

dehydrogenase (FadE14 or MbtN). FAAL33 activates and loads long-chain fatty

acids onto MbtL, which are then transferred by MbtK onto the €-amino group of

the core lysine. FadE14 is the enzyme responsible for the a,~- unsaturation present

on the acyl chain. The N-hydroxylation of the lysine €-amino group has been

shown to be catalyzed by the N6-hydroxylase, MbtG, from the mbtl locus by using

substrate mimics (Krithika et aI., 2006). This enzyme belongs to a class of

flavoprotein mono-oxygenase and uses molecular oxygen for hydroxylation.

Though the biosynthesis of mycobactin has been dissected out in details, the

complete sequence of events that leads to its assembly has not been elucidated.

1.4.2.9 PKS5 and PKS6

While PKS5 is a type I PKS with a domain organization of KS-AT-DH­

ER-KR-ACP, PKS6 is a type I PKS with a domain architecture of ACP-KS-A T­

ACP-TE, similar to PKS13 (Yadav et aI., 2003b). PKS5 is 66% identical to MAS

and biochemical analysis of a PKS5 mutant of Mtb reveals that its cell envelope

composition is identical to that of the wild type strain (Rousseau et aI., 2003b).

PKS5 could be involved in the biosynthesis of an unknown lipid which is a minor

constituent of the cell wall. PKS6, on the other hand, is implicated in the

biosynthesis of an unknown polar metabolite (Waddell et aI., 2005), and FAAL30

has been shown to be involved in the activation and transfer of starter fatty acyl

chains onto PKS6 (Trivedi et aI., 2004; Waddell et aI., 2005). Interestingly, a Mtb

- 75 -

Chapter 1

mutant of pks6 was impaired for growth in the lungs of Balb/c mice, suggesting

that the PKS6-derived metabolite may play an important role in mycobacterial

survival and virulence (Camacho et ai., 1999).

1.4.2.10 PKS14 and PKS16

PKS14 and PKS16 are wrongly annotated as PKSs in the H37Rv genome.

PKS 14 is a 120-amino-acid protein with no conserved domains and PKS 16 is a

544-amino acid protein belonging to the acyl activating super family of enzymes

(Cole et ai., 1998).

Mtb has clearly adopted novel biochemical mechanisms that facilitate its

survival under changing environmental conditions. Since the catalytic versatility

of PKSs is well recognized, it is not surprising that mycobacteria utilize these

enzymes for the biosynthesis of unique lipidic metabolites. The identification and

characterization of molecular mechanisms that generate functional diversity can

significantly expand our understanding of how this pathogen evades the host

immune system and survives under harsh conditions.

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