Natural Products in Chemical Biology (Civjan/Natural Products in Chemical Biology) || Modular Polyketide Synthases

7MODULAR POLYKETIDE SYNTHASES

Tonia J. Buchholz, Jeffrey D. Kittendorf, and DavidH. ShermanUniversity of Michigan, Ann Arbor, Michigan

Polyketides constitute a large class of microbial and plant-derived secondary metabo-lites that displays a vast array of structural diversity. These organic molecules varyin molecular weight, functional group modification, and include linear, polycyclic,and macrocyclic structural forms. Currently, polyketide natural products find clinicaluse as antibiotics, antiparasitic agents, antifungals, anticancer drugs, and immuno-suppressants. Given these impressive and wide-ranging pharmacologic activities, anever-increasing demand is placed on natural products research to uncover novelpolyketide metabolites for the benefit of human and animal health. Modular polyketidesynthases are nature’s platform for the expansion of chemical diversity. This reviewprovides new perspectives on important biosynthetic mechanisms that contribute tothis variety. This includes control of double-bond configuration and regiochemistry,introduction of β-branching during polyketide chain assembly, and other processesthat contribute to introduction of unique chemical functionality into these fascinatingsystems.

Despite the promise of modern synthetic technologies to enhance pharmaceuti-cal discovery significantly, natural products continue to be the greatest sourceof all new drug leads (1). Currently, many examples of natural product-derivedpharmaceuticals are employed to benefit human health (2, 3); polyketides con-stitute a large class of microbial and plant-derived secondary metabolites thatdisplays a vast array of structural diversity. These organic molecules vary inmolecular weight and functional group modification; they include linear, poly-cyclic, and macrocyclic structural forms. Currently, polyketide natural products

Natural Products in Chemical Biology, First Edition. Edited by Natanya Civjan.© 2012 John Wiley & Sons, Inc. Published 2012 by John Wiley & Sons, Inc.

163

164 MODULAR POLYKETIDE SYNTHASES

OH

O

O

O

O

O

OHHO

HOO

NMe2

O OH

OMe

MeO

O

O

O

O

O

N

OO

OH

HO

MeO

OMe

OH H

Erythromycin A • Saccharopolyspora erythraea • Target: bacterial ribosome • Antimicrobial

Epothilone B • Sorangium cellulosum • Target: microtubules • Antitumor

O

CH3O

O OH O

OH

S

N

Rapamycin • Streptomyces hygroscopicus • Target: mTOR kinase • Immunosuppressant

O

O

O

O

O

ON

O

OCH3

OHO

OH OH

O

O

O

OH

OH OH

O

Mycolactone • Mycobacterium ulcerans • Outcome: skin lesions/Buruli ulcer • Virulence Factor

Sulfolipid-1 • Mycobacterium tuberculosis • Potential virulence factor for tuberculosis

O

O

OO

O

O

OOH

O

HOHO

O3SO

O

O

O

OH

OH

RhizoxinBurkholderia sp.Target: β-tubulinOutcome: rice seedling blightAntimitotic Phytotoxin

(a)

(b)

Figure 7.1 Examples of clinically-relevant polyketide natural product drugs (a) andvirulence factors (b).

find clinical use as antibiotics, antiparasitic agents, antifungals, anticancer drugs,and immunosuppressants (Fig. 7.1a). Given these impressive and wide-rangingpharmacological activities, an ever-increasing demand is placed on natural prod-ucts research to uncover novel polyketide metabolites for the benefit of humanhealth.

Although the clinical use of polyketide-inspired pharmaceuticals has beenappreciated for decades, polyketide-derived metabolites have been recognizedrecently for their role in bacterial virulence. For example, the pathogenesis ofMycobacterium ulcerans , the causative agent of the devastating skin diseaseknown as Buruli ulcer, is the result of the secretion of polyketide-derived toxins

PROTOTYPICAL POLYKETIDE BIOSYNTHESIS 165

known as the mycolactones (Fig. 7.1b) (4). These polyketide toxins are respon-sible largely for the necrotic lesions that are characteristic of this debilitatingcondition. As such, the disruption of mycolactone biosynthesis may lead to aneffective chemotherapy for Buruli ulcer. Recent findings also suggest that thevirulence of another mycobacterial species, Mycobacterium tuberculosis , couldbe partially dependent on polyketide biosynthesis. The cell surface sulfolipid-1(SL-1) is among several virulence-associated molecules produced by M. tuber-culosis . SL-1 consists of a sulfated disaccharide core (trehalose-2-sulfate) thatdisplays four lipidic substituents; all but one substituent seems to be polyketide-derived (Fig. 7.1b) (5). Finally, the toxic agent in rice seedling blight, which is ahighly destructive fungal disease that inflicts severe agricultural losses worldwide,has been identified recently as the polyketide metabolite, rhizoxin (Fig. 7.1b) (6).Interestingly, rhizoxin is not produced directly by the fungus (Rhizopus), butrather by the endosymbiotic bacteria Burkholderia that thrives within the fungus.Together, these three examples suggest that inhibition of polyketide biosynthe-sis may lead to effective chemotherapy for controlling certain human and plantbacterial diseases.

7.1 PROTOTYPICAL POLYKETIDE BIOSYNTHESIS

The biosynthesis of many important polyketide compounds occurs via a stepwise,assembly-line type mechanism that is catalyzed by type I modular polyketidesynthases (PKSs). These modular PKSs are composed of several large, multi-functional enzymes that are responsible for catalyzing the initiation, elongation,and processing steps that ultimately give rise to the characteristic macrolactonescaffold (Fig. 7.2) (7–11). Structural studies have been critical in developing asophisticated understanding of the overall architecture and mechanism of type IPKSs and their homologs in recent years (12–18). A review from the perspec-tive of the 6-deoxyerythronolide B synthase (a well-studied type I PKS) waspublished recently by Khosla et al. (19).

It is well established that the sequential arrangement of modules within a PKSsystem serves effectively as a biosynthetic program, which is responsible fordictating the final size and structure of the polyketide core. Typically, initiationof polyketide biosynthesis begins by the acyltransferase (AT) catalyzed linkageof a coenzyme A (CoA) priming unit (e.g., methylmalonyl-CoA, malonyl-CoA,propionyl-CoA) to the acyl carrier protein (ACP) of the loading module. Onceinitiated, downstream elongation modules carry out repetitive extensions of thestarter unit. In most PKS systems, each elongation module contains at minimuman AT domain, an ACP domain, and a ketosynthase (KS) domain (Fig. 7.2a).The AT domain is responsible for loading the appropriate CoA extender unitonto the ACP domain (i.e., malonyl-CoA, methylmalonyl-CoA, etc.). The KSdomain then, catalyzes a decarboxylative condensation of the extender unit withthe growing polyketide chain obtained from the preceding module to gener-ate an ACP-bound β-ketoacyl product. In addition to the three core domains,


AT KR

DH ERACP

SH

KS

AT KR

DH ERACP

SH

KS

Ketosynthasecondenses 2-C extender

onto growing chain

Acyltransferaseselects extender unitand loads it onto ACP

Ketoreductasereduces β-ketone

to β-hydroxyl

Dehydratasedehydrates β-hydroxyl

to α-β double bond

Enoyl reductasereduces α-β double bond

to alkane

Acyl carrier protein

Phosphopantetheinealong with ACP, delivers

acyl chain to each catalytic site

C-terminal docking domainmediates inter-polypeptide

communication

N-terminal docking domainmediates inter-polypeptide

communication

TE - Thioesterase * - Inactive domain

OCoAS

O

OH

Starter and extender units

SNH

NH

OP

OP

OOO

OHO

OHO

N

N

N

N

H2N

O O O O

PO32

CoAS = O

CoAS

Methylmalonyl-CoA

1

35791113

Pikromycin PKS System

AT AT AT AT AT AT ATKS KS KS KS KS

KR KR KR KR

TE

DHER

ACP ACP ACP ACP ACP ACP

OS S

O

O

OH

OH

O

S

O

OH

OH

OS

O

OH

OS

O

OH

OS

O

OH

S

OH

O

loadmodule 1 module 3 module 5

module 6module 4module 2

end

PikAI PikAIIIPikAII

narbonolide

KR*

KSQ

DH

PikAIV

O

O

O

OH

O

O

O

O

OH

10-deoxymethynolide(10-dml)

1 3

5

7

9

11

13

O

OH

ACP KS

Malonyl-CoA

(a)

(b)

Figure 7.2 Conventional modular type I PKS paradigm. (a) Individual domains in afull type I polyketide synthase extension module. Homodimeric contacts are made in theN-terminal docking, ketosynthase, dehydratase, enoyl reductase, and C-terminal dockingdomains. (b) PKS system for 10-deoxymethynolide and narbonolide generation.

each elongation module may contain up to three additional domains [ketoreduc-tase (KR), dehydratase (DH), enoyl reductase (ER)] that are responsible for thereductive processing of the β-keto functionality prior to the next extension step(Fig. 7.2a). These reductive steps contribute greatly to the overall structural diver-sity that is observed among polyketide natural products. The presence of a KRdomain alone generates a β-hydroxyl functionality, the presence of both a KRand a DH domain generates an alkene, whereas the combination of KR, DH, andER results in complete reduction to the alkane. Finally, termination of polyketide

POLYKETIDE DOUBLE BONDS 167

biosynthesis is catalyzed by a thioesterase (TE) domain located at the carboxyterminus of the final elongation module. The activity of this domain results inthe cleavage of the acyl chain from the adjacent ACP; typically, intramolecularcyclization results in the formation and release of a macrolactone ring. Tailoringenzymes, such as hydroxylases and glycosyl transferases, often serve to furthermodify the polyketide to yield the final bioactive compound.

The modular organization of type I PKSs has made them particularly attractivetargets for rational bioengineering. Combinatorial biosynthetic efforts centeredon prototypical modular PKSs have been the topic of many recent outstand-ing review articles (20–23). Currently, several strategies are being pursued thatattempt to leverage PKS systems for the generation of structurally diverse polyke-tides. For example, it has been demonstrated that alterations of individual catalyticdomains (i.e., inactivation, substitution, addition, deletion) within a PKS modulecan result in predicted structural alterations of the final PKS product. Like-wise, the addition, deletion, or exchange of intact modules can also impartstructural variety into polyketide metabolites. Using these and other approaches,hundreds of novel polyketide structures have been generated, which establishedthe tremendous potential of these applications. However, these successes seemto be more the exception rather than the rule, as many efforts result in tracelevels, or they fail to provide the desired metabolite. This finding suggeststhat much remains to be learned regarding the molecular intricacies of thesecomplex biosynthetic machines. This review provides new perspectives on impor-tant mechanisms that contribute to structural diversity in modular PKSs. Thesemechanisms include control of double-bond configuration and regiochemistry,introduction of β-branching during polyketide chain assembly, and other pro-cesses that contribute to introduction of unique chemical functionality into thesefascinating systems.

7.2 POLYKETIDE DOUBLE BONDS

7.2.1 Trans Double Bonds

The presence of unsaturated carbon–carbon bonds within most polyketide com-pounds exemplifies the overall structural diversity that is a hallmark of this classof important natural products. Typically, the installation of double bonds intonascent polyketide chains relies on the two-step processing at the β-keto groupby the successive activity of KR and DH domains that are embedded within agiven PKS elongation module. After KS catalyzed chain elongation that extendsthe growing chain by two carbon atoms, the KR domain, when present, directsthe NADPH-dependent reduction of the β-ketone to yield a 3-hydroxyacyl inter-mediate. Subsequently, an embedded DH domain within the elongation modulecatalyzes dehydration of the 3-hydroxyacyl intermediate, normally which resultsin the incorporation of an (E)-trans α,β unsaturated bond into the growing polyke-tide chain (Fig. 7.3a).


(a)

(b)

(c)

AT KR

ACPKS

O

OH

S

O

O

OH

-O

O

O

TE

hydrolysis

thioesterase

decarboxylation

dehydration

??

TmcBModule 10

AT KR

ACPKS ST

hydrolysis

thioesterase?

decarboxylation

desulfurylation

??

CurM

TE

HO

O

S sulfuryl transfer

sulfotransferaseAT KR

ACPKS ST TE

CH O

O

O

S

S

O

O

O O

O

S

O

-O

O

-O

R

HO

OOH OH

R =

R R

R R R R

R =

N

SH

H H

10

3CH O

3 CH O3

CH O3

-

S

O O

ACPR

KS

AT

S

O O

R S

O

R

DH ER KR

S

OH O

RS

O O

R

KR

S

O O

R S

O

R

DH KR

ACP

ACP

ACP

ACP

ACP

ACP

Figure 7.3 (a) Traditional view of reductive processing at the β-ketone position in thegrowing polyketide chain. Presence of a ketoreductase domain leads to formation of analcohol. An active dehydratase domain can further process the alcohol moiety to an alkene.Complete saturation to the alkane is accomplished by an enoyl reductase domain. (b) Pro-posed terminal double bond formation for curacin biosynthesis. (c) Proposed terminaldouble bond formation in tautomycetin biosynthesis.

It should be noted that the KR catalyzed reduction of a β-ketoacyl intermediatehas stereochemical consequences because a new chiral center is introduced intothe growing oligoketide. Aside from serving to enhance the structural diversityof the final polyketide product even more, the stereochemical outcome of thisreaction can have profound effects on any subsequent processing or elongation


reactions. As such, an understanding of how KR domains exert stereochemi-cal control of their hydroxylated product is a critical aspect to deciphering themechanism of DH-mediated double bond formation. Through bioinformatic andbiochemical analyses, an appreciation of ketoreductase-influenced stereochem-istry has emerged (24, 25). Thus, Caffrey (25) has proposed that KR domainscan be divided into two classes, depending on the final configuration of theβ-hydroxyl moiety. The so-called “A” class generates an L-3-hydroxy prod-uct, whereas the “B” class produces the D-3-hydroxy polyketide intermediate.Although little difference exists between these two putative classes at the aminoacid sequence level, the presence of a conserved aspartate residue within anLDD motif correlates well with “B” class. This motif is absent in the defined“A” class of KR domains. An additional diagnostic feature of the “A” class of KRdomains is the presence of a conserved tryptophan residue. Recently, Keatinge-Clay (18) has proposed a refinement of the KR class descriptions as originallysuggested by Caffrey (25), effectively increasing the number of possible KR typesfrom two to six (18). This new classification takes into consideration whether agiven KR domain (either reductively competent or incompetent) is located in anepimerization-competent module. Although this new classification offers a morecomplete description of PKS KR domains, for simplicity we will continue to useCaffrey’s KR nomenclature throughout our discussion of double-bond formation.

While examples of both D- and L-hydroxyl group configurations can be foundwithin polyketide natural products, recent evidence suggests that DH domainsrequire a stereospecific 3-hydroxyacyl intermediate. Bioinformatic analyses per-formed on 71 KR domains for which the stereochemical outcome of the reductionis cryptic because of subsequent dehydration revealed that all belong to the “B”class of KR domains (25). As such, it appears that the generally preferred sub-strate for DH domains is a D-3-hydroxyacyl chain. However, direct experimentalevidence has been difficult to obtain because the 3-hydroxyacyl intermediate istransient in modules that contain a DH domain. Recently, biochemical studies ofthe DH domain found in module 2 of the pikromycin PKS system (Fig. 7.2b)(26) have supported this hypothesis; inactivation of the DH domain resulted inthe exclusive generation of the D-3-triketide acylthioester intermediate from adiketide substrate (27). Aside from this study, no other reports probe the sub-strate preference or catalytic mechanism of DH domains within PKS systems;therefore, much of what is known has been elucidated from studies of fatty acidbiosynthesis (28). Previous studies on the dehydration step that is catalyzed bythe yeast fatty acid synthase confirmed the syn elimination of water from a D-(3 R)-hydroxyacylthioester substrate (29). This result is consistent with the stere-ospecificity of the PKS DH domain and may suggest that trans unsaturated bonds,typically found in polyketides, are likewise formed via syn water elimination.

7.2.2 Cis Double Bonds

Although rare, several PKS biosynthetic systems can install cis double bondsinto the final polyketide product (Fig. 7.4). Several possible mechanisms could


Br

HN

ClO OCH

O N

O

7 - Jamaicamide

O O

H2O PO

4 - Difficidin

NS

H

H H

3 - Curacin A

OCH3

15

1310

4 3

18

20

2 - Bryostatin 1

H3CO O

O O

OHO

HO

O

O O

O

OCH3

O

O

HO

HO

OH

H

H

H

21

138

18

8 - Leinamycin

O

S

S S

O

O O

OH

OH

NH

N

6

3

1

O

HN

OH

OHN

O

HO

HO

1 - Bacillaene

1

9

1’

4’6’

12’

9’

OOH O

NO

OOH

O

N

O

O

OCH3

5 - Disorazole A1

O

O OOH

OH

S

N

6 - Epothilone D

11 12

1312

3

3

Figure 7.4 Double bond containing compounds discussed in this review. Cis-doublebonds discussed in the text are boxed and modifications produced by the HMGS cassettesare shaded.


O

O

NH

O

OH

O

N

O

O

N

15 - Virginiamycin M

O

OHO

HO

OH

O

O

OH

O

9 - Mupirocin

OHN

OOH

O OCH3

O

OCH3

OCH3

OH

12 - Pederin

OHN

OOH

O O

O

O

HO

NH

O COOH

NH

NH

H2N

11 - Onnamide

10 - Myxovirescin A

OH

HN

O

OH

OH

O

O

OCH3

O

16

12

OOOOH

H2N

O HOP(OH)2O

13 - Phoslactomycin

O

O

OO OHOHOH

OO

O

O

14 - Tautomycetin

23

15

12

OCH3

Figure 7.4 (Continued )

account for the infrequent occurrence of this double bond configuration. Oneexplanation is that an isomerization event occurs that converts a trans doublebond into a cis double bond. This isomerization activity could be specified bythe PKS elongation module, much like previously identified epimerization activ-ities that are known to exist in some PKS and NRPS modules. Alternatively,the combined activity of KR-DH domains within certain modules could directlyestablish the cis double bond. Finally, it is possible that after reduction of the β-keto functionality, a trans acting DH could catalyze dehydration to form a (Z )-cisdouble bond. This trans activity might derive from a discrete enzyme encoded


within the biosynthetic gene cluster or from an adjacent module within the PKSpathway. Additionally, examples exist in which some general rules may not holdtrue. Chivosazol, which is a potential antitumor agent, is one such example (30).

The epothilones 6 (and Fig. 7.1a), which are produced by Sorangium cellu-losum , are mixed NRPS-polyketide derived natural products that possess potentantitumor activity. Interestingly, some compounds feature a cis double bondbetween carbon atoms 12 and 13 that should be generated by PKS elongationmodule 4; however, sequence analysis of the epothilone biosynthetic gene clusterindicates that module 4 does not contain a DH domain requisite for the forma-tion of the unsaturated bond (31). Thus, Tang et al. (31) hypothesized that theDH activity might occur from the subsequent module or by the action of a post-PKS modifying enzyme. Biochemical experiments later demonstrated that the DHdomain of module 5 catalyzed the cis double bond formation (32). To accountfor this atypical activity, it is proposed that the 3-hydroxythioester intermedi-ate undergoes an ACP4-to-ACP5 transfer (32). After dehydration, the thioesterintermediate would then be transferred to the KS5 domain for subsequent elon-gation. Similarly, the antitumor phoslactomycin compounds 13 feature three cisdouble bonds, two of which seem to be installed by a KR-DH pair (see below);however, the elongation module (Plm7) that should be responsible for generatingthe unsaturated bond between carbon atoms 2 and 3 does not appear to encodethe required DH activity (33). Thus, it is likely that the source of this catalyticactivity comes from either a different module within the PKS system or a sep-arate enzyme that could act either before or after TE mediated termination ofpolyketide biosynthesis. Additional analysis is required to discriminate betweenthese two possibilities.

Unlike the examples described above, most polyketide cis double bonds areinstalled through a successive KR-DH pair found embedded within the elonga-tion module. In these cases, the stereochemistry of the 3-hydroxyacyl intermediateappears to be the discriminating factor between (Z )-cis or (E )-trans unsaturation.For example, the antitumor disorazole compounds 5 display up to three cis dou-ble bonds per monomer (note: final compound is a condensed dimer). Sequenceanalyses of KR domains preceding DH domains that would be responsible for cisdouble bond formation suggest that they all belong to the “A” class (34). Thus,they are predicted to generate a L-3-hydroxyacyl intermediate. It is expected thatthe subsequent DH domain preferentially recognizes the L-3-hydroxyl group tofacilitate the generation of the cis double bond. Interestingly, the module respon-sible for incorporating the cis unsaturated bond between carbon atoms 11 and12 is split between two polypeptide chains (34). It cannot be ruled out that thismodular dissection may play a role in formation of this particular double bond,as the cleavage point occurs between the DH and KR domain. Furthermore, it isintriguing that the major product, disorazole A1, is composed of two noniden-tical monomers that differ in saturation between carbon atoms 5 and 6. It hasbeen suggested that the synthesis of the two different monomers is caused bypoor activity of the DH domain (34); however, final proof requires additionalexperimental verification.


In addition to disorazole 5, several other known examples of polyketide natu-ral products exist that have cis double bonds installed by an embedded KR-DHdomain pair. The potent antitumor compound curacin A 3 contains many interest-ing structural features. Among them is the presence of a cis double bond betweencarbon atoms 3 and 4. Sequence alignment of the KR domain encoded by curG(encoding the module responsible for generation of the cis double bond) sug-gests that it belongs to the “A” class of KR domains (35). Thus, this particularKR is predicted to generate a L-3-hydroxyacyl intermediate that is subsequentlydehydrated to the cis double bond. Likewise, the KR domains that set up the3 cis double bonds found in the linear mixed NRPS-polyketide natural prod-uct bacillaene 1 are predicted to produce L-3-hydroxyacyl intermediates (36).Interestingly, two of the elongation modules that incorporate cis olefins are splitbetween two polypeptides (36). As in disorazole biosynthesis, it is possible thatthese modular dissections contribute to the configuration of the unsaturated bondthat is introduced by these modules.

Finally, we consider the conjugated cis olefins that span carbon atoms 12-15of the phoslactomycins 13. Analysis of the KR sequences of elongation modules1 and 2 could not clearly predict whether these reductive domains belonged tothe “A” or “B” class. Therefore, Alhamadsheh et al. (37) employed a compre-hensive biochemical study to elucidate the mechanism of cis olefin formation bythe first elongation module. Two hypotheses were considered. First, the config-uration of the double bond could develop directly from the combined activitiesof the embedded KR-DH domain pair. Alternatively, the KR-DH domains mightestablish a trans olefin that is isomerized subsequently to the observed cis con-figuration. To distinguish between these two possibilities, Alhamadsheh et al.(37) genetically inactivated both the loading module and elongation module ofPlm1 and conducted feeding experiments with diketide analogs containing bothcis and trans olefins. Results from this work indicated clearly that only thecis olefin containing diketide is accepted as a substrate for elongation module2, suggesting that the product of module 1 must contain the cis double bond.Furthermore, this work demonstrated nicely that the phoslactomycin biosyn-thetic pathway cannot process trans diketide intermediates into mature prod-ucts, which rules out the possibility of an isomerization domain in downstreammodules.

7.2.3 Terminal Double Bonds

Termination of polyketide biosynthesis typically involves the TE mediated cleav-age of the ACP-bound thioester, followed by cyclization to generate a macro-lactone. Alternatively, the TE catalyzes the simple hydrolysis of the thioester togenerate a linear free acid product. Here, we consider two of the relatively fewknown examples of polyketide natural products that are neither a macrocycle nora free acid, but instead terminate with a double bond.

Aside from containing a cis double bond noted above, the antitumor polyketidecompound curacin A 3 also features a terminal olefin. Previously reported feeding


studies suggested that the formation of the terminal double bond develops fromsuccessive decarboxylation and dehydration events (35). The biosynthetic genecluster responsible for curacin A biosynthesis has been identified and initiallycharacterized, which enables the putative assignment of domains within the pre-dicted elongation modules (35). Like most other known polyketide biosyntheticpathways, the final elongation module of the curacin pathway, CurM, containsa terminal thioesterase domain that presumably plays a role in formation of theterminal olefin; however, biochemical evidence for such a role is lacking. Inter-estingly, domain analysis of CurM also predicts the presence of a sulfotransferase(ST) domain immediately preceding the TE domain. Typically, ST domains areresponsible for transferring a sulfuryl group from a donor molecule (such as3′-phosphoadenosine-5′-phosphosulfate, PAPS) to a variety of acceptor carbo-hydrates, proteins and other low-molecular weight metabolites (38). AlthoughSTs have been characterized from both eubacterial and eukaryotic organisms, thepresence of an ST domain within a PKS system is unprecedented.

Current efforts in our laboratory include elucidating the roles of the ST andTE domains in curacin A biosynthesis, and in particular, their potential functionsin terminal olefin formation. One possible mechanism that is currently underconsideration is shown in Fig. 7.3b. On reduction of the β-keto functionality ofthe ACP-bound thioester intermediate, the ST domain transfers a sulfuryl groupfrom the donor molecule PAPS to the 3-hydroxyl group of the thioester chain.Consistent with this hypothesis, bioinformatic analysis suggests that the putativecuracin ST domain contains the signature PAPS binding pocket (unpublisheddata); however, no experimental evidence suggests that this ST domain can cat-alyze the sulfuryl transfer or that the ST domain can bind the requisite PAPSdonor molecule. Assuming our hypothesis is correct and that the ST domainfunctions as proposed, transfer of the sulfurylated intermediate to the TE domainwould initiate hydrolytic termination of curacin A biosynthesis to produce thelinear free acid. At this point, one of several chemical steps can be envisioned.Following hydrolysis, the TE may catalyze decarboxylation, after which the for-mation of the double bond would occur in a concerted process by displacementof the sulfate leaving group. Alternatively, a separately encoded enzyme mightbe responsible for decarboxylating the free acid generated by the TE domain. Itis also conceivable that on TE catalyzed hydrolysis, the decarboxylation reactionoccurs spontaneously due to the presence of the sulfate leaving group at carbon 3.

Similar to curacin, the polyketide metabolite tautomycetin 14 also possesses aterminal olefin. This polyketide metabolite has potential medicinal value becauseof its novel immunosuppressive activities (39). The tautomycetin biosyntheticgene cluster has been sequenced recently, which enables domain compositionanalysis of the terminal elongation module (40). Unlike the terminal moduleinvolved in curacin biosynthesis, the final tautomycetin elongation module doesnot contain the unusual ST domain. This finding may suggest that formation ofthe terminal olefin of tautomycetin 14 occurs by a different chemical mechanism.The final elongation module does contain a TE domain, which presumably termi-nates tautomycetin biosynthesis through generation of the free acid (Fig. 7.3c).

ATYPICAL PKS DOMAINS 175

As described for curacin, it is possible that this TE domain can also catalyzethe subsequent decarboxylation event; however, in lieu of an activated leav-ing group at carbon 3, it is reasonable to expect that dehydration to removethe hydroxyl at carbon 3 would also be a catalyzed event. Alternatively, theterminal olefin could be installed during the post-PKS maturation of the polyke-tide to the final tautomycetin product. DNA sequence analysis of open read-ing frames that are downstream of the tautomycetin PKS gene cluster revealstwo potential candidates, tmcJ and tmcM , which may be involved in doublebond formation (40). Bioinformatic analyses suggest that tmcJ might encodefor a putative decarboxylase and that the gene product of tmcM is a potentialdehydratase.

7.3 ATYPICAL PKS DOMAINS

The wide distribution of PKSs in the microbial world and the extreme chemicaldiversity of their products do in fact result from a varied use of the well-knowncatalytic domains described above for the canonical PKS systems. Taking a the-oretic view of polyketide diversity, Gonzalez-Lergier et al. (41) have suggestedthat even if the starter and extender units are fixed, over 100,000 linear hep-taketide structures are possible using only the 5 common reductive outcomes atthe β-carbon position (ketone, (R- or S -) alcohol, trans-double bond, or alkane).Recently, it has become apparent that even this does not represent the upperlimit for polyketide diversification. To create chemical functionalities beyondthose mentioned above, nature has recruited some enzymes from sources otherthan fatty acid synthesis (the mevalonate pathway in primary metabolism is oneexample) not typically thought of as type I PKS domains. Next, we explore theways PKS-containing systems have modified these domains for the catalysis ofsome unique chemistries observed in natural products.

7.3.1 Methyl Groups at the α- and β-carbons

As described above for polyketide biosynthesis, the presence or absence ofa methyl group on the α-carbon position of the growing polyketide chain ismost often governed by the selection of the extender unit (malonyl-CoA versusmethylmalonyl-CoA). However, in PKS systems that use trans acyltransferases(AT-less type I PKSs) (8, 42), the module by module control over extender unitselection is sometimes not possible. In most cases, malonyl-CoA is used as theextender unit, and a methyl group can be added to selected positions through theaction of an embedded methyl transferase (MT) domain or discrete MT enzyme.For example, the C-6 methyl group of leinamycin 8 is thought to be installed bythe MT embedded in LnmJ (43), the C-10 methyl of curacin A 3 is generatedvia the MT domain in CurJ (35), and the gem dimethyl groups on C-8 and C-18of bryostatin 2, most likely are the consequence of the MT domains in BryB andBryC (44).


In contrast to the α-carbon methylations, the incorporation of methyl or methy-lene groups (or functional groups derived from such groups) at the β-positionrepresents the assimilation of a full cassette of enzymes into the typical PKSmachinery. Recently, a subset of type I modular PKSs (and hybrid NRPS/PKSmegasynthases) have been identified that contain multiple enzymes acting in transduring the traditional linear assembly-line process to accomplish β-branching.Termed HMG-CoA synthase (HMGS) cassettes, these enzyme systems providea unique method of expanding the repertoire of the traditional reductive domains(KR, DH, ER). These enzymes work in conjunction with the PKS machineryto create unique functionalities observed at the branch points that include thependant methyl groups of bacillaene 1 (36, 45, 46), mupirocin 9 (47), andvirginiamycin M 15 (48), which are the methoxymethyl and ethyl groups ofmyxovirescin A 10 (49, 50); the exo methylene groups of difficidin 4 (45),onnamide A 11 (51), and pederin 12 (51–53); the cyclopropyl ring of curacin A3 (35, 54); the vinyl chloride of jamaicamide 7 (55); the unique 1,3-dioxo-1,2-dithiolane moiety of leinamycin 8 (43); and the exocyclic olefins in bryostatin 2(Fig. 7.4) (44).

7.3.2 HMG-CoA Synthase Cassettes

In primary metabolism, HMG-CoA synthase (HMGS) is responsible for thecondensation of C-2 of acetyl-CoA onto the β-ketone of acetoacetyl-CoA toform 3-hydroxyl-3-methylglutaryl-CoA and free CoASH (Fig. 7.5a) (56). Sev-eral secondary metabolite pathways have been identified over the past five yearsthat perform an analogous reaction, although they seem to use ACP-tetheredacyl groups (Fig. 7.5b) as opposed to acyl-CoA substrates. After generation ofthe HMG-ACP analog on the growing polyketide chain, the product is usu-ally dehydrated and decarboxylated to yield the branched intermediate. Foundin 11 pathways to date (36, 45–55), included in the cassette are a discreteACP, a decarboxylative KS (active site cysteine is replaced with a serine), anHMGS, and one or two enoyl CoA hydratase-like (ECH) domains. (Table 7.1,Figs. 7.5, 7.6).

7.3.3 HMGS Cassette Biochemistry

Three HMGS-containing cassettes (those in the curacin A, bacillaene, andmyxovirescin pathways) have been validated biochemically and will serveas the basis for our analysis of the individual components in this complex(46, 54, 57, 58). The mechanistic and structural details for HMG-CoAsynthase in primary metabolism have been elucidated for both bacterial andeukaryotic HMGSs (59–63). Although polyketide HMGSs share only 20–30%sequence identity with their primary metabolism homologs (in both prokaryotesand eukaryotes), multiple sequence alignment reveals that the key catalyticresidues (Glu/Cys/His) are conserved. As shown in Fig. 7.5b, the first step in

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the formation of the HMG-intermediate is the generation of of acetyl-ACP. Thisstep is accomplished through the loading via an AT of malonyl-CoA [or perhapsmethylmalonyl-CoA in at the case of TaE (58)]. Then, the decarboxylative KSconverts the malonyl-ACP into acetyl-ACP, after which the tethered acetyl groupis condensed onto the β-ketone of the polyketide intermediate. Finally, formationof the HMG-analog is completed on addition of water.

Processing of the HMG-intermediate can vary considerably, but typicallyproceeds via dehydration and decarboxylation catalyzed by two enoyl-CoAhydratase-like domains (Fig. 7.5b). Based on sequence similarity, the membersof the crotonase fold family observed in these HMGS cassettes can be subdividedinto two groups, termed ECH1 and ECH2 (54). The successive dehydration anddecarboxylation steps are catalyzed by the ECH1 and ECH2 enzymes/domains,respectively. Evidence for the specific function of the curacin ECH1 and ECH2

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180

ATYPICAL PKS DOMAINS 181

converted first to 3-methylglutaconyl-ACP then to 3-methylcrotonyl-ACP, whichis the gained intermediate for subsequent formation of the cyclopropyl ring.Insights into the mechanism of the CurF ECH2 decarboxylation have been gainedbased on the recent crystal structure of the curacin ECH2 domain (64). Addi-tional in vitro evidence for the function of these enzymes has been generatedusing proteins from the PksX pathway of Bacillus subtilis (46) and the myx-ovirescin pathway from Myxococcus xanthus (58). Prior to the identificationof bacillaene as the product of the PksX pathway, Calderone et al. (46) andDorrestein et al. (57) reported the function of several discrete enzymes. Usingradioactive biochemical assays together with mass spectrometry, they assignedfunctional roles to AcpK, PksC, the tandem ACPs in PksL, PksF, PksG, PksH,and PksI. Using the model acceptor ACP, acetoacetyl-ACP, and malonyl-CoA incombination with the above proteins, a �2-isoprenyl-S-carrier protein was gen-erated (46). Most recently, a similar in vitro investigation was conducted usingthe homologous enzymes from the myxovirescin pathway (58). The HMGS cas-sette reaction sequence proposed above held fast for the myxovirescin pathway,although the generation of the propionyl- or methylmalonyl-S -ACP could not bedemonstrated. The authors have suggested that perhaps additional enzymes areyet to be identified to fill these roles to complete the β-ethylation at C16 in 10.Two variations on the HMGS cassette theme already have been identified. In thebiosynthesis of bryostatin 2, and leinamycin 8, one or both of the ECH-mediatedsteps is likely omitted based on the final natural product structures. The detailsof these deviations have not yet been established.

Recently, in vivo evidence for the function of these HMGS cassettes hascome from the Muller lab (49, 50, 65). To date, all HMGS cassette proteinsfor myxovirescin A (TaB/TaC, TaE/TaF, TaK, TaX, TaY) has been individuallydeleted, and the impact on the products of the engineered Myxococcus xanthusstrains has been analyzed. In addition, analysis of products from �taV (thetrans-acting AT), �taH (a cytochrome P450 that is thought to hydroxylatethe HMGS-installed β-methyl group at C12 of 10), and �taQ (an O-methyltransferase necessary for completing the transformation to the final methoxymethyl functionality) strains have provided insights into this complex pathway.Production of myxovirescin A was abolished (or greatly reduced) in all abovedeletion strains. Appearance of novel myxovirescin analogs (β-methyl vs β-ethylat C16) in the �taE & �taF strains appears to be a result of TaB or TaCcomplementation, which provides direct evidence for TaE/TaF in the formationof the ethyl branch point. However, independent biochemical verification of TaFfunction has been difficult to obtain (58).

7.3.4 HMGS Cassette Architecture

Analysis of the placement of the known HMGS cassettes identified to date intotheir biosynthetic clusters reveals a variety of possible architectures (Fig. 7.6).For example, the ECH2 decarboxylase exists as a discrete enzyme downstreamof the ECH1 dehydratase (mupirocin and others), as an N-terminal domain of


a large PKS (curacin and jamaicamide), and as an embedded domain (pederinand onnamide). Although most clusters published to date are mixed PKS/NRPSsystems with in trans ATs and tandem ACPs at the site of HMGS modifica-tion, exceptions exist for each example (difficidin is PKS only, curacin andjamaicamide contain embedded ATs, and bryostatin and myxovirescin do notcontain tandem ACPs at the site of HMGS modification).

As HMGS enzyme cassettes have been identified and functionally character-ized only recently, many mechanistic details as well as the key protein–proteininteractions needed to orchestrate communication among the polypeptide com-ponents remain unclear. Details on how the individual proteins are brought tothe correct place in the pathway to perform their functions are still unknownfor most pathways. In the case of the PksX/bacillaene pathway, some intrigu-ing microscopy performed on B. subtilis suggests that the bacillaene proteinsare clustered into a huge mega-enzyme factory inside the bacterial cell (66).Whether this organization extends (or is limited) to the other members of HMGScassette containing pathways remains to be observed. Additionally, in some path-ways, key enzymes have yet to be identified. Two lingering questions include, 1)Which AT domain loads the discrete ACP in the embedded AT systems typifiedby the curacin and jamaicamide pathways? and 2) where are the missing domainslocated in the incomplete cassettes? Despite these remaining issues, the stage isnow set for these unique suites of enzymes to be included and applied in thegrowing metabolic engineering/combinatorial biosynthesis toolbox.

The goal of this review has been to highlight a series of novel systems for cre-ating chemical diversity in polyketide natural product biosynthesis. This reviewincludes the mechanistic basis for introduction of trans or cis double bondswithin linear or macrocyclic compounds, or assembly of the rare terminal alkenein select secondary metabolites such as curacin and tautomycetin. Similarly, intro-duction of methyl groups to create branch points or gem dimethyl functionalitycan occur by several processes that have been dissected in several systems overthe past few years. Finally, one of the most intriguing new methods for introduc-tion of diverse branching functionality involves the HMGS-containing enzymesthat are being identified in a growing number of PKS and mixed NRPS-PKSpathways. The rapidly increasing knowledge and mechanistic understanding ofthese complex metabolic systems will provide growing opportunities to engineerchemical diversity using rational approaches.

7.4 ACKNOWLEDGMENTS

Research on modular type I polyketide synthases in the Sherman laboratoryis supported generously by grants from the National Institutes of Health(GM076477, CA108874, TW007404), the Hans and Ella McCollum VahlteichResearch Fund at the University of Michigan College of Pharmacy, and theH.W. Vahlteich Professorship in Medicinal Chemistry. J.D.K. is supported byan NRSA postdoctoral fellowship (GM075641) from the NIH.

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Natural Products in Chemical Biology (Civjan/Natural Products in Chemical Biology) || Modular Polyketide Synthases