Programming of ErythromycinBiosynthesis by a ModularPolyketide Synthase*Published, JBC Papers in Press, June 3, 2010, DOI 10.1074/jbc.R110.144618

David E. Cane1

From the Department of Chemistry, Brown University,Providence, Rhode Island 02912-9108

Since the discovery and sequencing of 6-deoxyerythronolide Bsynthase 20 years ago, this exceptionally large, multifunctionalprotein remains the paradigm for the understanding of the struc-ture and biochemical function of modular polyketide synthases.The broad-spectrummacrolide antibiotic erythromycin is one ofseveral hundred closely related, branched chain, polyoxygenatedpolyketides, many of which are widely used in human and veteri-narymedicine as antibiotic, immunosuppressant, antitumor, anti-fungal, and antiparasitic agents. The multistep assembly of theparent macrocyclic aglycon, 6-deoxyerythronolide B (6-dEB),2 iscontrolled by a large (2 MDa) modular protein known as 6-dEBsynthase (DEBS) (1–4). The biosynthetic intermediates neverleave thepolyketide synthase (PKS) but are passed along theDEBSassembly line from one acyl carrier protein (ACP) domain to thenext. In fact, DEBS has served as the prototype of modular PKSgene clusters, dozens of which of both known and unknown func-tion have now been sequenced from bacterial sources (5–7).Each homodimeric DEBS subunit contains two 160–200-kDa

protein modules, each responsible for a single round ofpolyketide chain extension and functional group modification.Within each module are several catalytic domains of 100–400amino acids each that are analogous in structure, function, andorganization to the corresponding fatty acid synthase (FAS)components (8–10). All six DEBS modules contain three coredomains consisting of 1) a �-ketoacyl-ACP synthase (the keto-synthase (KS) domain) that catalyzes the key polyketide chain-building reaction, a decarboxylative condensation of a methyl-malonyl-ACP building block with the polyketide chainprovided by the upstream PKSmodule (see Figs. 1 and 2); 2) anacyltransferase (AT) domain that specifically loads the methyl-malonyl-CoA extender unit onto the flexible 18-Å phospho-pantetheine arm of the ACP domain; and 3) the ACP domainitself, which carries the polyketide biosynthetic intermediatesfrom domain to domain and then delivers the resulting productto the KS domain of the downstreammodule. Additional FAS-like domains are responsible for modification of the oxidation

state and stereochemistry of the growing ACP-bound interme-diates: a�-ketoacyl-ACP reductase (KR) domain, a dehydratase(DH) domain, and an enoyl reductase (ER) domain. At the Nterminus of the most upstream module is a loading didomainthat primes the KS domain of module 1 with the propionylstarter unit. Finally, cyclization of the full-length macrocyclicpolyketide and release of 6-dEB are controlled by a dedicatedthioesterase domain located at the C terminus of the mostdownstream module.

Programming of Polyketide Biosynthesis

The chain length, substitution pattern, and oxidation level ofthe initially generated, full-length heptaketide 6-dEB are thedirect consequence of the number of DEBS modules as well asthe domain composition of each module (Fig. 1) (8–11). Thestriking colinearity between the organization of the constituentbiosynthetic domains encoded by theDEBS genes and the orderof the biochemical reactions that generate 6-dEB had originallyencouraged the hope that the sequence of any newly discoveredPKSwould allow the ready prediction of the chemical structureof the derived polyketide product. Unfortunately, such straight-forward correlations have proven to be elusive. Unlike the uni-directionally transcribed DEBS cluster, a variety of modularPKS genes are either divergently or convergently transcribedsuch that the sequential order of the open reading frames foundin the genome does not correspond to the temporal order inwhich they exercise their biochemical function (12). Recentprogress in understanding the structural basis for pairwiseinteractions of the docking domains that direct the properintermodular transfer of polyketide intermediates may allowbetter correlations of deduced sequences and temporal func-tion (13–16). Further obscuring an overly simplistic linearinterpretation is the presence in individual PKS modules ofinactive DH or ER domains, whereas an additional complica-tion comes fromPKSmodules that lack integrated AT domainsaltogether and therefore require the in trans AT-catalyzedpriming ofACPdomainswith the requisite extender units. Bro-ken modules in which the domains mediating a single round ofchain elongation are located in adjacent PKSmodules, aswell asthe phenomena of module skipping or iteration (17), furtherobscure the relationship of PKS gene sequence and biochemicalfunction. The existence of unusual polyketide-building blockssuch as methoxymethylmalonyl-CoA and hydroxymethylma-lonyl-CoA and the recognition of polyketide chain branchinghave added additional layers of biochemical complexity (18).Despite promising new bioinformatics approaches (15), knowl-edge of the specific structure of the eventually formed poly-ketide has therefore turned out to be critical to the properassignment of PKS organization and function. Nonetheless, thewell studiedmolecular genetics and biochemistry of DEBS con-tinue to provide the dominant paradigm for the understandingof modular PKS biochemistry (17).The 14-member ring of 6-dEB displays an impressive aggre-

gate of 10 stereogenic centers. Similar stereochemical patternsare found in a wide range of closely related 12-, 14-, and

* This work was supported, in whole or in part, by National Institutes of HealthGrants GM22172 (to D. E. C.) and CA87394 (to C. Khosla). This is the thirdarticle in the Thematic Minireview Series on Antibacterial Natural Products.This minireview will be reprinted in the 2010 Minireview Compendium,which will be available in January, 2011.

1 To whom correspondence should be addressed. E-mail: david_cane@brown.edu.

2 The abbreviations used are: 6-dEB, 6-deoxyerythronolide B; DEBS, 6-dEB syn-thase; PKS, polyketide synthase; ACP, acyl carrier protein; FAS, fatty acid syn-thase; KS, �-ketoacyl-ACP synthase; AT, acyltransferase; KR, �-ketoacyl-ACPreductase; DH, dehydratase; ER, enoyl reductase; NDK-SNAc, natural diketide(2S,3R)-2-methyl-3-hydroxypentanoyl-N-acetylcysteamine thioester.

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16-member ring macrolides (19, 20). Only recently has themechanistic basis for the remarkable stereochemical complex-ity of the polyketides generated by DEBS and other modularPKS proteins begun to be unraveled.

PKS Modules and Domains

Heterologous expression of engineered PKS modules hasplayed a central role in the elucidation of the biochemicalmechanisms, substrate specificity, protein-protein recognitionfeatures, and structures of DEBSmodules and domains (21, 22).DEBS modules, including those with inactivated, mutated, ordeleted domains, have been expressed in both native and het-erologous bacterial hosts, with the structure and stereochemis-try of the resulting polyketide products serving as a readout ofthe role of individual modules or domains (21–23). Recent pro-gress in the development of large deletion mutants of Strepto-myces avermitilis as hosts for the heterologous expression ofbiosynthetic gene clusters (24) should provide a powerful newtool for the assignment of biochemical function to cryptic PKSgene clusters and the discovery of new polyketides.Individual modules or domains can be expressed using both

native and heterologous Streptomyces hosts as well as strains ofEscherichia coli harboring a chromosomally integrated copy ofthe sfp gene from Bacillus subtilis that encodes the surfactinphosphopantetheinyl transferase (25) necessary to convert theACP domains to their active form (26). Investigations of thespecificity and function of PKS domains have frequently uti-lized chimeric modules harboring engineered combinations ofdomains derived from a variety of parent modules (22, 27–29).The labor-intensive construction of suchhybrid systemshas forthe most part relied on domain boundaries deduced frommultiple sequence alignments. Unfortunately, the resultinghybrids have often suffered from a severe loss in overallcatalytic efficiency compared with wild-type modules.Recently, detailed structural studies of PKS modular compo-nents have revealed highly conserved functional domainboundaries that can now be exploited for the expression

of individual active recombinantPKS domains (30–32). Not onlyare these stand-alone domains ex-cellent vehicles for protein struc-tural and mechanistic study, butreconstituted mixtures of thesePKS domains allow investigationof reaction mechanisms, protein-small molecule specificity, and therole of protein-protein recogni-tion in the programming and con-trol of PKS function.

KS Domains

The irreversible KS-catalyzedchain elongation reaction involvesthe initial formation of a covalentacyl-enzyme intermediate betweenthe growing polyketide chain, do-nated by the ACP of the upstreammodule, and the active-siteCys thiol

of the KS, followed by a decarboxylative condensation of thisacyl thioester with methylmalonyl-ACP (33, 34) with inversionof configuration at C2 of the (2S)-methylmalonyl moietyto produce the corresponding D-2-methyl-3-ketoacyl-ACPintermediate (Fig. 2) (35). Eventual generation of the L-methylconfiguration found at C8 and C12 of 6-dEB requires an epi-merization reaction at some stage subsequent to KS-catalyzedchain elongation. Besides the active-site Cys (36), each KSdomain harbors a pair of universally conservedHis residues (forexample, His334 and His374 in DEBS KS5) that facilitate thedecarboxylation and condensation reactions (37, 38). (2S,3R)-2-Methyl-3-hydroxypentanoyl-N-acetylcysteamine thioester(NDK-SNAc), the N-acetylcysteamine acyl thioester analog ofthe natural ACP-bound syn-diketide substrate of DEBSmodule2, can conveniently be used as a surrogate substrate for DEBSKS domains (39, 40).

Structures of the DEBS [KS5][AT5] and [KS3][AT3]Didomains

The 2.7-Å x-ray crystal structure of a 194-kDa homodimericKS and AT didomain of DEBSmodule 5, [KS5][AT5], providedthe first direct insights into the topology and three-dimensionalorganization of a modular PKS (Fig. 3a) (31). These findingshave been reinforced by the 2.6-Å x-ray crystal structure of thehomologous 190-kDa homodimeric [KS3][AT3] didomainfrom DEBS module 3 with the inhibitor cerulenin covalentlybound to the active-site Cys of the KS3 domain (32). In bothstructures, the homodimeric KS domains and the monomericATdomains at the end of each protein arm closely resemble thestructures of previously characterized type II FAS- and PKS-derived KS and AT domains. The [KS5][AT5] homodimer car-ries the N-terminal docking domain with the homodimericcoiled-coil structure previously deduced frommutant comple-mentation and NMR studies (13, 14). Although the truncated[KS3][AT3] construct lacks the corresponding N-terminaldocking domain, twowell organized interdomain linker regionsare apparent in both structures: 1) a highly ordered KS-to-AT

FIGURE 1. Modular organization of DEBS. In addition to the KS, AT, and ACP domains, individual extensionmodules carry varying combinations of KR, DH, and ER domains. The loading didomain primes DEBS module 1with the propionyl starter unit, and the thioesterase (TE) domain at the C terminus of module 6 catalyzes releaseand cyclization of the full-length polyketide to give the parent macrolide aglycon, 6-dEB. Two dedicated P450oxygenases, two glycosyltransferases, and a methyltransferase then generate the mature antibiotic, erythro-mycin A. (2S)-MeMal-CoA, (2S)-methylmalonyl-CoA.

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linker domain with a previously uncharacterized protein foldand 2) a 30-amino acid post-AT linker appended to the C ter-minus of the AT domain that wraps back over both the ATdomain and the KS-to-AT linker and terminates on the face ofthe KS domain distal to the N-terminal docking domain. Therelative orientations of the paired AT and KS domains and ofthe post-AT linker support earlier observations that KS andACP domains from opposite subunits of a homodimeric

module preferentially interact (36,41). Computational docking haspredicted that the ACP should dockto the KS domain within the deepcleft between the KS and AT do-mains, with interactions that spanacross both subunits of the KShomodimer (32). The two crystalstructures also revealed that theactive-site Cys199 of the KS is sepa-rated from the active-site Ser642 ofthe nearest AT domain by �80 Å.This distance is obviously too greatto be spanned by a statically an-chored, fully extended 18-Å phos-phopantetheine arm of the ACPdomain. Thus, the interaction of theACP domain with each of the cata-lytic domains of a modular PKSmust involve substantial segmentalmotion of the entire ACP domain,rather than simple reorientation ofthe pantetheinyl prosthetic group,contrary to the classical “swingingarm” model of PKS and FAS action.Similar conclusions have beendrawn from the 3.2-Å structure ofthe complete pig FAS protein (42,43). The similarity of these two struc-tures suggests a model for the entireDEBS module 4 with an analogousX-shaped architecture (Fig. 3e) (2).The recombinant DEBS [KS1]-

[AT1], [KS3][AT3], and [KS5][AT5]didomains have been used to studypolyketide chain elongation in com-bination with both homologous andheterologous ACP domains (30, 44).Each [KS][AT] didomain plus anACP domain can be incubated withNDK-SNAc and methylmalonyl-CoA to give a triketide ketolactoneafter hydrolytic release (Fig. 4a).The [KS3][AT3] and [KS6][AT6]didomains both exhibit a dis-tinct ACP recognition profile, withstrong preference for the ACPdomain from their parent modules.Using chemoenzymatically pre-pared methylmalonyl-ACP and

malonyl-ACP as substrates, KS3 has a 4:1 preference for itsnative methylmalonyl extender unit, whereas KS6 shows asmaller 1.5:1 preference. These findings highlight the key gate-keeping role of the AT domains that must charge the ACPwiththe correct chain extender unit while demonstrating the intrin-sic ability of KS domains to utilize unnatural chain extensionunits. The reconstituted DEBS domain mixtures allow quanti-tative kinetic comparisons that are not possible using intact

FIGURE 2. Biochemical function of individual PKS domains, illustrated by polyketide chain extension andfunctional group modification mediated by the domains of DEBS module 4. The AT domain loads the(2S)-methylmalonyl group onto the pantetheinyl side chain of the ACP domain, whereas the KS domain cata-lyzes self-acylation of its active-site Cys residue by the tetraketide chain donated by the upstream ACP domain.The KS domain then catalyzes a decarboxylative condensation to generate a D-2-methyl-3-ketoacyl-ACP inter-mediate, which then undergoes successive KR-catalyzed reduction, DH-catalyzed dehydration, and ER-cata-lyzed reduction to the reduced pentaketide, which is then transferred to the KS domain of DEBS module 5.Configurational assignments are based on the known or predicted stereochemistry of individual PKS reactions.

FIGURE 3. Structures of DEBS catalytic domains. a, DEBS [KS5][AT5] didomain, including the homodimeric KSdomain (light blue and dark blue), AT domains (green), N-terminal docking domain (orange-brown), KS-ATlinkers (yellow), and post-AT linkers (red). b, DEBS KR1, with the structural domain (cyan), catalytic domain (blue),and Leu-Asp-Asp (LDD) loop. c, DEBS DH4, with the double hot dog fold and catalytic His-Asp dyad. d, DEBSapo-ACP2. e, predicted organization and topology of DEBS module 4, analogous to the structure of porcineFAS. Each ACP domain (not shown) must be able to interact with the individual KS, AT, KR, DH, and ER domains.ppant, phosphopantetheine; act, active.

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chimeric modules (45). The addition of native or foreign C-ter-minal docking domains to the ACP domains has negligibleeffect on intramodular KS-ACP recognition during KS-cata-lyzed condensation. By contrast, intermodular self-priming ofDEBS KS(n) domains by acyl donors carried on upstreamACP(n�1) domains is relatively insensitive to overall ACPstructure but strongly dependent on a proper match of thecomplementary C-terminal ACP(n�1) and N-terminal KS(n)docking domains.The [KS3][AT3] didomain can also be dissected into its com-

ponent KS3 and AT3 domains (46). The recombinant AT3domain, either with or without the appended post-AT linker,can catalyze both self-acylation by exogenous methylmalonyl-CoA and transfer of this methylmalonyl moiety to the ACP3domain. Similarly, the dissected KS3 domain undergoes self-catalyzed acylation by NDK-SNAc. By contrast, formation ofthe triketide ketolactone by KS-catalyzed chain elongationrequires that the added AT3 domain carry an appendedpost-AT linker. The extensive interaction between thispost-AT linker and the KS domain that is observed in both the[KS3][AT3] and [KS5][AT5] crystal structures therefore mustplay a critical functional role in proper alignment of the KS andAT domains and productive interaction with the ACP-boundsubstrate.

AT Domains

The AT domain of each DEBS module plays a special role asgatekeeper, being strictly specific for (2S)-methylmalonyl-CoA(22, 47, 48). The universally conserved active-site Ser in eachDEBS AT domain, found in a signature GHSQGEmotif, is partof a canonical catalytic dyad in partnership with the conservedactive-site His. The active-site His is believed to act as a generalbase that activates nucleophilic attack by the Ser hydroxyl onthe acyl group of the methylmalonyl thioester and then assistsin the transfer of the methylmalonyl-O-Ser intermediate to thephosphopantetheinate side chain of the ACP domain (Fig. 2).

The AT domain contains an �/�-hydrolase-like core domain and anappended smaller subdomainwith aferredoxin-like structure (Fig. 3a)(31, 32). TheAT domains from eachof the six DEBS modules are com-pletely specific for (2S)-methylma-lonyl-CoA (47). By contrast, the ATdomain in the AT-ACP loadingdidomain strongly prefers propio-nyl-CoA but can also use acetyl-CoA to prime the paired ACPdomain (49).

KR Domains

The DEBS KR domains, in com-mon with all other PKS and FAS KRproteins, belong to the large familyof short chain dehydrogenase/re-ductases, all of which harbor a con-served active-site Ser-Tyr-Lys triadresponsible for binding and activa-

tion of the target carbonyl group of the �-ketoacyl-ACP sub-strate while simultaneously orienting and activating the nico-tinamide ring of the NADPH cofactor (50). The stereochemicalcourse of each �-ketoacyl-ACP reduction is an intrinsic prop-erty of the responsible KR domain and is independent of eithermodular context or substrate structure, including chain lengthand substitution pattern (51). DEBS KR1, KR2, KR5, and KR6all utilize the H4si hydride of the NADPH cofactor (Fig. 2), incommonwith FASKR domains (52, 53). A conserved Leu-Asp-Asp triad found inmany PKSKR domains is strongly correlatedwith the generation of D-hydroxy products (54, 55). Crystalstructures of the DEBS KR1 domain and the homologous KR1domain from module 1 of tylactone synthase have indicatedthat the loop harboring this Leu-Asp-Asp motif may play a keyrole inmediating proper access and orientation of the phospho-pantetheinyl-bound �-ketoacyl thioester substrate in the com-mon substrate-binding groove (Fig. 3b) (56, 57). In KR domainssuch asDEBSKR2, KR5, andKR6, which lack this Leu-Asp-Aspmotif but harbor instead a conserved Trp at an alternativesite, the phosphopantetheinyl-bound �-ketoacyl substrate isthought to be constrained to enter the substrate-bindinggroove from the opposite end, giving rise to the observed L-hy-droxy product. Keatinge-Clay (57) and Starcevic et al. (58) havealso proposed that the redox-inactiveDEBSKR30 domainsmaybe responsible for the epimerization of the 2-methyl group togive the L-2-methyl-3-ketoacyl-ACP tetraketide intermediateproduced by DEBS module 3.The solution of the 1.8-Å structure of the DEBS KR1 domain

also resulted in a major redefinition of the KR domain bound-aries that had been previously erroneously inferred solely frommultiple sequence alignments (56). Each monomeric KRdomain consists of two principal subdomains: an N-terminalstructural subdomain and aC-terminal catalytic subdomain, eachwith a modified Rossmann fold (Fig. 3b). The structural subdo-main, which is thought to stabilize the catalytic subdomain, lacks

FIGURE 4. Stereochemistry of DEBS KR-catalyzed reduction and methyl group epimerization using mix-tures of individual recombinant domains and didomains and gas chromatography-mass spectrometryanalysis of the derived triketide lactones (a) and diketide acids (b).

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the characteristicGXGXXGNADPH-bindingmotif butmay pro-vide a docking site for the ACP domain (56, 57).

Stereochemistry of KR-catalyzed 3-Ketoacyl-ACPReductions

The reduction of the 2-methyl-3-ketoacyl-ACP substrate bya KR domain also fixes the configuration of the 2-methyl sub-stituent. Recombinant DEBSKR1 reduces racemic 2-methyl-3-ketopentanoyl-SNAc exclusively to a syn-(2S,3R)-2-methyl-3-hydroxy diketide (NDK-SNAc), thereby demonstrating thatthis reductase is completely specific not only for reduction onthe re-face of the ketone carbonyl but also for the natural L-con-figuration of the adjacent 2-methyl group, consistent with theoverall stereospecificity of DEBS module 1 (59, 60).Co-incubation of recombinant DEBS KR2 or KR6 plus

NADPH with reconstituted DEBS [KS1][AT1] � ACP1,[KS3][AT3]�ACP3, or [KS6][AT6]�ACP6 in the presence ofNDK-SNAc and methylmalonyl-CoA gives the correspondingreduced triketide lactone (Fig. 4a) (61, 62). DEBSKR2,KR5, andKR6 are completely diastereoselective, generating a productwith the identical (2R,3S)-2-methyl-3-hydroxy stereochemistryas that of the parent DEBS modules from which they had beenderived (Figs. 1 and 4a). Reductive quenching withNaBH4 con-firmed that DEBS [KS1][AT1], [KS3][AT3], and [KS6][AT6] allgenerated exclusively the unepimerized D-2-methyl-3-keto-acyl-ACP triketide intermediate (61, 62). Unexpectedly, thisACP-bound 2-methyl-3-ketoacyl triketide thioester was con-figurationally remarkably stable, undergoing �5–15% epimer-ization even after 1 h, �15–45-fold slower than the measuredrate for buffer-catalyzed deuterium exchange of untetheredmethyl-2-methyl acetoacetate. This remarkable enhancementin configurational stability is thought to be due to sequestrationof the bound polyketide in the ACP cleft between helices 2 and3 and consequent conformational restriction that significantlyreduces the acidity of the H2 proton of the 3-ketoacyl-ACPsubstrate.When propionyl-SNAc is used as the primer for DEBS

[KS1][AT1], [KS3][AT3], or [KS6][AT6] and their cognateACPdomains, DEBS KR1 generates only the reduced and epimer-ized (2S,3R)-L-2-methyl-D-3-hydroxypentanoyl-ACP product(62). Alternatively, incubation with DEBS KR6 gives the pre-dicted (2R,3S)-diketide acid. For each reconstituted incubationmixture, the absolute and relative stereochemistry of eachchain elongation and reduction product is strictly correlatedwith the intrinsic hydroxyl and methyl group stereospecificityof the particular DEBSKR domain, independent of whether theKS domain utilized is derived from a module that normallyproduces a reduced unepimerized product (DEBS KS6), anunreduced epimerized product (DEBS KS3), or a reducedepimerized product (DEBS KS1). These results are all consis-tent with KR1-catalyzed epimerization following KS-catalyzedchain elongation. The mechanism of this epimerization,including the amino acid residues that are responsible for thisprocess, is currently unknown.

DH Domains

Although the vast majority of polyketides generated bymod-ular PKSs contain one or more double bonds, in DEBS only

module 4 harbors a DH domain. The transiently generatedenoylacyl-ACP pentaketide produced byDEBSDH4 is ordinar-ily not observed due to coupled reduction by the paired ER4enoyl reductase domain. Disruption of the NADPH-bindingmotif of this ER4 domain in the complete DEBS proteinresulted in the accumulation of a derivative of the correspond-ing (E)-�6,7-anhydro-6-dEB (63). The DH4-catalyzed dehydra-tion is expected to take place with syn-stereochemistry by anal-ogy to the known stereospecificity of the DH domain of yeastFAS (Fig. 2) (64). The DEBS DH4 domain carries a conservedHXXXGXXXXP motif that carries part of the established His-Asp catalytic dyad, in which the active-site His acts as a generalbase to remove the C2 proton, whereas the Asp donates a pro-ton to promote departure of the 3-hydroxyl group (65). Indeed,the H2409F mutant of DEBS module 4 did not produce 6-dEB(66). The 1.85-Å structure of recombinant DEBS DH4 displaysthe double hot dog fold also observed in the DH domain of pigFAS (Fig. 3c) (67). The active-site His and Asp dyad of DEBSDH4 can be accessed by a tunnel leading from the surface of theprotein adjacent to a proposed ACP-binding region.

ER Domains

The ER domain of DEBS module 4 is inserted between thestructural and catalytic subdomains of the KR4 domain. TheDEBS ER4 domain reduces its unsaturated pentaketide sub-strate by net conjugate addition of a hydride from NADPH toC3 of the E-enoylacyl-ACP, with addition of a proton to C2.Disruption of the putative NADPH-binding motif of ER4,2964HAAAGGVGMA, by the double mutant HAAASPVGMAabolishes enoyl reduction (63). An active-siteTyr influences thestereochemistry of the reduction (68).

ACP Domains

The DEBS ACP domains serve as carriers of the growingpolyketide chain, which is covalently tethered as the acyl thio-ester to the phosphopantetheinyl prosthetic group that is inturn attached to the universally conserved serine found withinthe LGXDSmotif of each ACP. It is now evident that the ACPdomains of modular PKSs and FASs must undergo consider-able segmental motion to access the active sites of the individ-ual domains (31, 32, 42, 43, 69). The NMR solution structure ofthe 10-kDa recombinant DEBS apo-ACP2 reveals a three-helixbundle connected by two loops, with an additional short helixin the second loop also contributing to core helical packing (Fig.3d) (70). The overall protein surface of DEBS ACP2 appears tobe less charged comparedwith the type II FAShomologs, whichare more highly acidic. Homology models for each of the fiveremaining DEBS ACP domains have been calculated (70).Although the overall topology is largely conserved, there aremuch greater differences in the calculated electrostatic poten-tial surfaces, which may account for the observed discrimina-tion in functional interaction of DEBS ACP domains with theircognate KS domains.

Unanswered Questions

Twenty years after the sequencing of the 6-deoxyerythrono-lide synthase, we now have a broad and deep understanding ofthe fundamentals of the molecular enzymology and structural

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biology of DEBS and related modular PKS megaenzymes.Numerous unanswered questions remain. 1)What is themech-anistic basis for the KR-catalyzed methyl group epimerizationsthat take place during the formation of 6-dEB and otherpolyketides? 2)What is the balance between substrate specific-ity and protein-protein recognition in the programming ofpolyketide biosynthesis? 3) What are the structural and dy-namic details of the interaction of KS acceptor domains andupstreamACP donor domains? 4)What is the structural basis forthe interaction of ACP domains with the constituent KS, AT, KR,DH, and ER domains of each module, and what are the detaileddynamics by which charged ACP domains find and interact witheach�-carbon-processing domain? 5)What is themolecular basisfor programming and specificity of type I PKSs that vary from thecanonicalDEBSparadigm?Unraveling theworkingsof thesecom-plex and fascinating syntheticmachineswill ultimately require thedevelopment of new physical-biochemical techniques and thecontinued collaboration of chemists, biochemists, microbialgeneticists, and structural biologists.

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David E. CaneProgramming of Erythromycin Biosynthesis by a Modular Polyketide Synthase

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