Enzyme Discovery for Natural Product Biosynthesis Hongnan Cao1, Fengbin Wang1, Mitch M. Miller1, Weijun Xu1, Ragothaman M. Yennamalli1, Lu Han1, Joey Olmos1, Craig Bingman2, Kate Helmich2, Michael G. Thomas2, Jeremy R. Lohman3,5, Ming Ma3, Jeffrey, Rudolf3, Ben Shen3, Shanteri Singh4, Jon S. Thorson4,

Sherif I. Elshahawi4, Midwest Center for Structural Genomics, Northeast Center for Structural Genomics, George N Phillips Jr.1* 1Rice University, Houston, TX, 2University of Wisconsin-Madison, 3The Scripps Research Institute, FL, 4University of Kentucky, 5Purdue University, IN (current),. *georgep@rice.edu

The Center for Natural Product Biosynthesis (NatPro) was established in July 2011 with the funding from the National Institute of General Medical Sciences (NIH) entitled Enzyme Discovery for Natural Product Biosynthesis. The goals of NatPro are to apply technologies developed by the Protein Structure Initiative (PSI) to problems of interest to the community of biologists and biochemists who investigate the role of natural products in human health and disease. We have determined over 60 enzyme structures from multiple antitumor antibiotic biosynthetic pathways..

NatPro plays strong joint roles in both the identification of new natural product pathways and the subsequent discovery of new natural product-based lead compounds by revealing the structures and active sites of novel enzymes, characterizing the enzymatic reactions of the gene products, identifying new natural products, and thus offering opportunities to identify and customize the pathways by altering specificities and/or identifying novel proteins or domains with desired enzymatic properties. The range of structures described includes glycosyltransferases, enzymes for methylation, polyketide synthase domains and other enzymes with novel enzymatic activities.

Acknowledgement This work is funded by Natural Product Biosynthesis (NatPro) grant U01GM098248 from National Institute of Health/National Institute of General Medical Sciences (NIH/NIGMS), NIH grant CA78747 and BioXFEL grant NSF 1231306. We would like to thank staff at the GM/CA beamline at the Advanced Photon Source for help and advice on data collection. We acknowledge all the beamline scientists and use of APS synchrotron, funded by DOE.

C-1027 (9-membered enediyne from Streptomyces globisporus )

S-Adenosylmethionine synthetase (Sulfolobus solfataricus) Biosynthesis of enediyne antitumor antibiotics Biosynthesis of Leinamycin (Streptomyces atroolivaceus)

SgcE6 flavin reductase (PDB 4R82; Tan, et al. 2014)

SgcC phenol monooxygenase (PDB 4OO2; Cao, et al. 2014) SgcE10 ACP-polyene thioesterase

(PDB 4I4J; Kim, et al. 2012)

Epoxide hydrolase (PDB 4I19; Tan, et al. 2012)

SgcJ with unknown function (PDB 4OVM; Chang, et al. 2013)

Neocarzinostatin (9-membered enediyne, from Streptomyces carzinostaticus)

Calicheamicin (10-membered enediyne, from Micromonospora echinospora )

CalG3 glycosyltransferase (PDB 3OTI)

CalS11 TDP-rhamnose 3’-O- methyltransferase (PDB 3TOS)

CalS8 TDP-glucose dehydrogenase (PDB 4XR9; Michalska et al. 2015)

CalU16 self-sacrifice resistance protein (PDB 4FPW) Elshahawi, et al. (2014) ACS Chem. Biol. 9, 2347

Biosynthesis of the enediyne antitumor antibioticC-1027 involves a new branching pointin chorismate metabolismSteven G. Van Lanen*, Shuangjun Lin*, and Ben Shen*†‡§

*Division of Pharmaceutical Sciences, †University of Wisconsin National Cooperative Drug Discovery Group, and ‡Department of Chemistry,University of Wisconsin, Madison, WI 53705

Edited by Christopher T. Walsh, Harvard Medical School, Boston, MA, and approved November 27, 2007 (received for review September 14, 2007)

C-1027 is an enediyne antitumor antibiotic composed of fourdistinct moieties: an enediyne core, a deoxy aminosugar, a !-aminoacid, and a benzoxazolinate moiety. We now show that thebenzoxazolinate moiety is derived from chorismate by the sequen-tial action of two enzymes—SgcD, a 2-amino-2-deoxyisochoris-mate (ADIC) synthase and SgcG, an iron–sulfur, FMN-dependentADIC dehydrogenase—to generate 3-enolpyruvoylanthranilate(OPA), a new intermediate in chorismate metabolism. The func-tional elucidation and catalytic properties of each enzyme aredescribed, including spectroscopic characterization of the productsand the development of a fluorescence-based assay for kineticanalysis. SgcD joins isochorismate (IC) synthase and 4-amino-4-deoxychorismate (ADC) synthase as anthranilate synthase compo-nent I (ASI) homologues that are devoid of pyruvate lyase activityinherent in ASI; yet, in contrast to IC and ADC synthase, SgcD hasretained the ability to aminate chorismate identically to thatobserved for ASI. The net conversion of chorismate to OPA by thetandem action of SgcD and SgcG unambiguously establishes a newbranching point in chorismate metabolism.

2-amino-2-deoxyisochorismate dehydrogenase !2-amino-2-deoxyisochorismate synthase ! 3-enolpyruvoylanthranilate

C -1027 is an enediyne antitumor antibiotic that is one of themost cytotoxic natural products ever discovered (1). It is

isolated from Streptomyces globisporus as a noncovalent apo-protein (CagA)–chromophore complex (2), with the chro-mophore consisting of four distinct covalently appended moi-eties: an enediyne core, a deoxy aminosugar, a !-amino acid, anda benzoxazolinate moiety (Fig. 1) (3). Although the enediynecore is directly implicated in the generation of a benzenoiddiradical that is capable of abstracting hydrogen atoms fromDNA leading to double-stranded DNA breaks (4), the othermoieties of C-1027 are critical in enhancing the bioactivity. Thisis clearly exemplified with the benzoxazolinate moiety, whichspecifically binds to CagA, hence providing stability to theenediyne core (5, 6), and is essential for both binding andintercalating the minor groove of DNA (7).

The biosynthetic precursor for the benzoxazolinate moiety ofC-1027 was initially proposed to be chorismate based on theuncovering of proteins homologous to anthranilate synthase(AS) components I and II that are located within the biosyntheticgene cluster (8). ASI, typically annotated as TrpE, catalyzes thefirst committed step in Trp biosynthesis—the conversion ofchorismate to anthranilate—whereas ASII (TrpG) provides theamine, using glutamate as the donor (9). In addition to the Trpbiosynthetic pathway, chorismate is also the branch point forminimally five other pathways found in primary and secondarymetabolism, the majority of which are initiated by ASI homo-logues (Fig. 2). This includes 4-amino-4-deoxychorismate(ADC) synthase (10), the first step in folate biosynthesis; iso-chorismate (IC) synthase (11, 12), the first step in enterobactin,pyochelin, and menaquinone biosynthesis; and salicylic acid (SA)synthase (13, 14), an enzyme family involved in the production

of the yersiniabactin and mycobactin. Two distinct chorismate-using families that have no significant sequence homology to ASIare also known; chorismate lyase (15), the first enzyme inubiquinone biosynthesis, and chorismate mutase (16), the firstenzyme in Phe and Tyr biosynthesis.

The reaction catalyzed by ASI is the composite of twoenzymatic steps: a reversible 1,5-substitution involving amineaddition at C2 of chorismate with concomitant loss of thehydroxyl group at C4 to yield trans-6-amino-5-[1-carboxyethe-nyl)oxy]-1,3-cyclo-hexadiene-1-carboxylic acid, commonlyknown as 2-amino-2-deoxyisochorismate (ADIC), followed byirreversible pyruvate elimination to form anthranilate. Theoverall reaction coordinate is known to proceed with ADIC asan enzyme bound intermediate, as deduced from (i) the con-version of synthetic ADIC to anthranilate (17, 18) and (ii)transient accumulation of small amounts of ADIC from aSalmonella typhimirium ASI (H398M) mutant (19). Althoughthe crystal structures for ASI (20–22), SA synthase (13, 23), ICsynthase (24), and ADC synthase (25) in combination withmechanistic studies on ASI, ADC synthase, and IC synthase(26–28) have provided clear insights into the nature of thenucleophile addition/elimination step (e.g., the ADIC synthaseactivity of ASI), the identification of the catalytic residuesessential for pyruvate lyase activity have remained enigmatic.For instance, although the ASI (H398M) mutant accumulated

Author contributions: B.S. designed research; S.G.V.L. and S.L. performed research; S.G.V.L.,S.L., and B.S. analyzed data; and S.G.V.L. and B.S. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.§To whom correspondence should be addressed. E-mail: bshen@pharmacy.wisc.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0708750105/DC1.

© 2008 by The National Academy of Sciences of the USA

Fig. 1. Structure of C-1027 with four distinct structural moieties.

494–499 ! PNAS ! January 15, 2008 ! vol. 105 ! no. 2 www.pnas.org"cgi"doi"10.1073"pnas.0708750105

were subjected to inactivation by gene dis-ruptions (see SOM). Inactivation of geneswithin the C-1027 gene cluster, as exempli-fied by sgcA, sgcC, sgcC1, sgcD6, and sgcE,abolished C-1027 production (Fig. 3), where-as that of genes outside the C-1027 genecluster, such as ORF(–5), ORF(–3), andORF54, had no effect on C-1027 production,leading to the assignment of the clusterboundaries at sgcB1 and sgcR3, respectively.C-1027 production was monitored by bioas-say against Micrococcus luteus and high per-formance liquid chromatography (HPLC)analysis of 1 (SOM), which undergoes facileBergman cyclization to yield the aromatized

product (3) (Figs. 1 and 3A) (13, 14). Theidentities of 1 and 3 were confirmed by elec-trospray ionization-mass spectrometry (ESI-MS) analyses. 1 showed (M ! H)! and (M !Na)! ions at m/z " 844 and 866, consistentwith the molecular formula C43H42N3O13Cl,and 3 showed an (M ! H)! ion at m/z " 846,consistent with the molecular formulaC43H44N3O13Cl. Consistent with the struc-ture of 1, those identified within the C-1027cluster include 13 genes, sgcE to sgcE11 andsgcF, encoding the enediyne core (4) biosyn-thesis; seven genes, sgcA to sgcA6, encodingdeoxy aminosugar (5) biosynthesis; sixgenes, sgcC to sgcC5, encoding #-amino acid

(6) biosynthesis; and seven genes, sgcD tosgcD6, encoding benzoxazolinate (7) biosyn-thesis (Fig. 2).

Three types of PKSs are known forpolyketide biosynthesis in bacteria. Type Iand type II systems are known for aliphatic(10) and aromatic polyketides (11), respec-tively, and type III system is largely knownfor monocyclic aromatic polyketides (15,16). Because the enediyne cores bear nostructural resemblance to any characterizedpolyketides, we cannot predict what type ofPKS is responsible for their biosynthesis. Infact, it remains controversial whether theenediyne cores are assembled by de novo

Fig. 2. Organization of the C-1027 biosynthesis gene cluster (A) andbiosynthetic hypothesis for the enediyne core and a convergent assemblystrategy (B), deoxy aminosugar (C), #-amino acid (D), and benzoxazolinate(E). Proposed functions for individual ORFs are summarized in GenBank

under accession number AY048920. ORFs outside the sgcB1 to sgcR3 regionare not essential for C-1027 production. Color coding indicates the biosyn-thesis genes for the enediyne core (red), deoxy aminosugar (blue), #-aminoacid (green), benzoxazolinate (purple), and all other genes (black).

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Chemistry & Biology294

identification of the NCS biosynthetic gene cluster fromS. carzinostaticus ATCC15944, the sequence of thecomplete NCS gene cluster, determination of the clus-ter boundaries, and functional assignments of the geneproducts. On the basis of these results, we propose aconvergent model for NCS chromophore biosynthesisfrom the deoxy aminosugar, naphthoic acid, and enedi-yne core building blocks. The NCS cluster is charac-terized with two distinct type I PKSs, NcsE for the ene-diyne core and NcsB for the naphthoic acid moiety, anda deoxysugar biosynthetic pathway most likely startingfrom dNDP-D-mannose. These findings further supportthe iterative type I PKS paradigm for enediyne core bio-synthesis [15–19], unveil a novel mechanism for micro-bial polycyclic aromatic polyketide biosynthesis by aniterative type I PKS [19, 20], and shed new light intodeoxysugar biosynthesis. The NCS cluster, togetherwith the growing list of other enediyne biosyntheticgene clusters [15–18], provides a unique opportunity toinvestigate the molecular basis of enediyne biosynthe-sis by a comparative genomics approach.

Figure 1. Structures of Neocarzinostatin (NCS) and C-1027 Chro-Results and Discussion mophores and Calicheamicin (CAL) γ1

I

Identification, Localization, and Cloning of the ncs resulted in the isolation of four overlapping cosmidsGene Cluster from S. carzinostaticus ATCC15944 spanning 67 kb (Figure S1B), the ncsA probe affordedDeoxysugars are frequently found in secondary metab- fourteen overlapping cosmids spanning 64 kb, as rep-olites and are vital components for the efficacy and resented by pBS5002, pBS5003, and pBS5004 (Figurespecificity of a natural product's biological activity. The 2A). However, the two loci do not overlap. Since genesbiosynthetic pathways of these unusual sugars have for antibiotic production are known to cluster in onebeen extensively investigated. The first committed step region of the chromosome in Streptomyces, we set outof the biosynthesis of all deoxyhexoses is through the to first determine if the putative NGDH locus was re-intermediate dNDP-4-keto-6-deoxyhexose, a reaction quired for NCS biosynthesis by gene disruption. Sur-catalyzed by an NAD+-dependent oxidoreductase from prisingly, inactivation of the NGDH gene has no effectthe precursor dNDP-D-hexose (most commonly dNDP- on NCS production, excluding its involvement in NCSD-glucose, and hence the name of NGDH derived from biosynthesis (Figure S1C).dNDP-D-glucose 4,6-dehydratase) [21]. We have pre- We next turned our attention to the ncsA locus. Sinceviously taken advantage of the highly conserved nature it was proposed that ncsA should reside within the NCSof NGDHs and used degenerate primers [22] to amplify gene cluster, a 7.5 kb BglII fragment containing ncsAthe NGDH gene sgcA by PCR from S. globisporus, and was cloned from pBS5004. DNA sequence analysis ofthis locus was used as a starting point for chromo- this fragment revealed six complete ORFs (includingsomal walking leading to the eventual localization of ncsA) and one incomplete ORF. Remarkably, the fourthe entire C-1027 biosynthetic gene cluster [15, 23]. ORFs encode a dNDP-D-mannose synthase (NcsC),NCS contains a similar 6-deoxyhexose to C-1027, and dNDP-hexose 4,6-dehydratase (NcsC1, a second dis-as a result, we adopted the same strategy to identify tinct NGDH gene in this organism), N-methyltransferasethe ncs gene cluster. A distinct product with the pre- (NcsC5), and glycosyltransferase (NcsC6). These aredicted size of 550 bp was amplified by PCR and con- the enzymes that would be predicted to be essential forfirmed to be a putative NGDH gene, serving as the first biosynthesis of the deoxy aminosugar moiety of NCSprobe (Figure S1A). chromophore (Figure 3A), indicating that the NCS bio-

The cloning of the genes necessary for C-1027 bio- synthetic locus was identified. To ensure that we havesynthesis revealed that cagA, encoding the C-1027 full coverage of the entire NCS gene cluster, additionalapo-protein, was within the boundaries of the gene chromosomal walking from the left end of pBS5002cluster [15]. Therefore, the gene for the NCS apo-pro- (probe 3) and the right end of pBS5004 (probe 4) wastein, ncsA, was used as the second probe. The primary carried out, leading to the eventual localization of a 130sequence for ncsA has previously been established [re- kb continuous DNA region covered by overlapping cos-viewed in 11], and PCR with primers designed accord- mids as exemplified by pBS5002, pBS5003, pBS5004,ing to the known sequence yielded a distinct product pBS5005, pBS5007, pBS5010, pBS5013, pBS5015, andwith the predicted size of 590 bp. The PCR product was pBS5017 (Figure 2A).cloned into pGEM-T to yield pBS5024, confirmed byDNA sequencing, and utilized as the second probe. Sequencing and Organization

The digoxigenin-(DIG)-labeled NGDH and ncsA of the ncs Gene Clusterprobes were both used to screen approximately 4800 Three representative overlapping cosmids, pBS5002,

pBS5004, and pBS5017 were selected for DNA se-clones of the genomic library. While the NGDH probe

Liu, et al. (2005) Chem. Biol. 12, 293

Chang, et al. (2011) PNAS USA. 108, 17649. Singh, et al. (2013) ACS Chem. Biol. 8, 1632

(AML) (6 ) points to therapeutic promisefor calicheamicin.

Although innovative synthetic schemeshave been devised for calicheamicin (7),surprisingly little is known about how thewarhead is synthesized in nature. Our under-standing of calicheamicin biosynthesis is lim-ited to a set of metabolic labeling experi-ments performed on the related enediyneesperamicin (Fig. S1, compound 5) (8). Wereport the elucidation of the calicheamicingene locus from Micromonospora echinos-pora ssp. calichensis, which reveals an un-usual iterative polyketide synthase (PKS)gene (calE8). Disruption of this PKS gene inthe calicheamicin-producer Micromonosporaprovides a strain no longer capable of pro-ducing calicheamicin and suggests that thisPKS is critical for enediyne formation. Ahomolog of this PKS has also been found inthe C-1027 gene cluster encoding a memberof the structurally distinct chromoproteinenediynes [see companion paper (9)]. In con-trast to previous biosynthetic hypotheses(10), this finding suggests that all enediynesmay share a common biosynthetic origin.This work also lays the foundation for futuremetabolic engineering strategies to generateenediyne analogs.

Using a combination of polymerase chainreaction (PCR)–based screening and screensfor clones capable of conferring calicheamicinresistance (11), nine overlapping cosmid cloneswere isolated from a genomic library of M.echinospora ssp. calichensis (NRRL 15839).Although sequence analysis of these clones wasconsistent with genes encoding aryltetrasaccha-ride biosynthesis and calicheamicin resistance,initial attempts to complete the biosyntheticlocus by chromosomal walking were unsuc-cessful (12). To circumvent this problem, weused a shotgun-based approach to finish thecalicheamicin locus. DNA sequence analysisrevealed 74 open reading frames (ORFs) thatspan more than 90 kb (Fig. 2). They include 16genes expected to participate in polyketide con-struction or modification (O1 to O6 and E1 toE10), nine putative regulatory elements (R1 toR9), seven genes associated with membranetransport (T1 to T7), 14 genes consistent withthe expected production of four unusual acti-vated nucleotide sugars (S1 to S14), four gly-cosyltransferase genes (G1 to G4), an insertion-al element (IS), and 22 ORFs (U1 to U22) ofunknown function (13). The known resistancegene calC (11) was also found to reside near themiddle of this locus.

Given the long-standing controversy sur-

rounding the biosynthetic origin of theenediyne moiety, the polyketide-associatedgenes within the calicheamicin gene clusterare most relevant to our hypothesis. The twodistinct calicheamicin structural elements—the calicheamicin warhead (Fig. 1, PKS E)and the orsellinic acid derivative found with-in the aryltetrasaccharide (Fig. 1, PKS O)—are each expected to derive from a separatepolyketide precursor. Consistent with this,the calicheamicin locus (Fig. 2, genes calE8and calO5) encodes two separate iterativetype I PKSs (CalE8 and CalO5). CalO5shows striking similarity (65% identity, 93%similarity) to the PKS (AviM) responsible fororsellinic acid biosynthesis in S. viridochro-mogenes en route to the biosynthesis of theorthosomycin antibiotic avilamycin A (14).Thus, CalO5 likely plays an identical role inthe biosynthesis of the calicheamicin aryltet-rasaccharide. In contrast, CalE8 demonstratesthe greatest sequence homology to two iter-ative PKSs involved in the biosynthesis of thepolyunsaturated fatty acids (PUFAs) eicosa-pentaenoic acid (EPA) in Shewanella (15,17) and docosahexaenoic acid (DHA) inMoritella marina (16, 17).

To confirm that CalE8 is critical to thebiosynthesis of calicheamicin in Micromonos-pora, a calE8 gene disruption was constructedby introduction of an apramycin selectablemarker (Fig. 3A). The calicheamicin producerM. echinospora is resistant to most antibiotics,and is inherently indisposed to genetic manip-ulation, with a best reported transformation ef-ficiency of 10!7 transformants per microgramDNA (18). In spite of these difficulties, a com-bination of many gene disruption attempts in M.echinospora LL6000 (19) led to nine indepen-dent apramycin-resistant clones. All nine iso-lates mapped consistently with the expectedcalE8 gene disruption by both PCR fragmentamplification and Southern hybridization. Therepresentative hybridization results from twoof these clones (pJAW117/LL6000-1 andpJAW117/LL6000-3) are illustrated in Fig.3B. All nine calE8 disruption mutants andtwo representative LL6000 isolates were sub-

Fig. 1. The structure of the nonchromoprotein enediyne calicheamicin. The enediyne families aretypically distinguished by either the number of carbons in the enediyne ring (9-membered versus10-membered) or by their association, or lack thereof, with an apoprotein (chromoprotein versusnonchromoprotein). With one exception (N1999A), the 9-membered enediynes are chromoproteinenediynes, whereas all 10-membered enediynes to date are nonchromoprotein. The colors correlatethe calicheamicin final structure to the functions of genes within the calicheamicin locus (Fig. 2)with the specific PKS structural components designated as PKS E and PKS O.

Fig. 2. The calicheami-cin locus from Mi-cromonospora echinos-pora spp. calichensis.The colors of genes de-lineate putative roles incalicheamicin biosyn-thesis, based on BLASTanalysis and correlateby color to thecalicheamicin structurepresented in Fig. 1. Thecorresponding calE8translation, CalE8, isalso shown to highlightthe location of themaindomains KS, AT, KR, DH,and TD.

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(AML) (6 ) points to therapeutic promisefor calicheamicin.

Although innovative synthetic schemeshave been devised for calicheamicin (7),surprisingly little is known about how thewarhead is synthesized in nature. Our under-standing of calicheamicin biosynthesis is lim-ited to a set of metabolic labeling experi-ments performed on the related enediyneesperamicin (Fig. S1, compound 5) (8). Wereport the elucidation of the calicheamicingene locus from Micromonospora echinos-pora ssp. calichensis, which reveals an un-usual iterative polyketide synthase (PKS)gene (calE8). Disruption of this PKS gene inthe calicheamicin-producer Micromonosporaprovides a strain no longer capable of pro-ducing calicheamicin and suggests that thisPKS is critical for enediyne formation. Ahomolog of this PKS has also been found inthe C-1027 gene cluster encoding a memberof the structurally distinct chromoproteinenediynes [see companion paper (9)]. In con-trast to previous biosynthetic hypotheses(10), this finding suggests that all enediynesmay share a common biosynthetic origin.This work also lays the foundation for futuremetabolic engineering strategies to generateenediyne analogs.

Using a combination of polymerase chainreaction (PCR)–based screening and screensfor clones capable of conferring calicheamicinresistance (11), nine overlapping cosmid cloneswere isolated from a genomic library of M.echinospora ssp. calichensis (NRRL 15839).Although sequence analysis of these clones wasconsistent with genes encoding aryltetrasaccha-ride biosynthesis and calicheamicin resistance,initial attempts to complete the biosyntheticlocus by chromosomal walking were unsuc-cessful (12). To circumvent this problem, weused a shotgun-based approach to finish thecalicheamicin locus. DNA sequence analysisrevealed 74 open reading frames (ORFs) thatspan more than 90 kb (Fig. 2). They include 16genes expected to participate in polyketide con-struction or modification (O1 to O6 and E1 toE10), nine putative regulatory elements (R1 toR9), seven genes associated with membranetransport (T1 to T7), 14 genes consistent withthe expected production of four unusual acti-vated nucleotide sugars (S1 to S14), four gly-cosyltransferase genes (G1 to G4), an insertion-al element (IS), and 22 ORFs (U1 to U22) ofunknown function (13). The known resistancegene calC (11) was also found to reside near themiddle of this locus.

Given the long-standing controversy sur-

rounding the biosynthetic origin of theenediyne moiety, the polyketide-associatedgenes within the calicheamicin gene clusterare most relevant to our hypothesis. The twodistinct calicheamicin structural elements—the calicheamicin warhead (Fig. 1, PKS E)and the orsellinic acid derivative found with-in the aryltetrasaccharide (Fig. 1, PKS O)—are each expected to derive from a separatepolyketide precursor. Consistent with this,the calicheamicin locus (Fig. 2, genes calE8and calO5) encodes two separate iterativetype I PKSs (CalE8 and CalO5). CalO5shows striking similarity (65% identity, 93%similarity) to the PKS (AviM) responsible fororsellinic acid biosynthesis in S. viridochro-mogenes en route to the biosynthesis of theorthosomycin antibiotic avilamycin A (14).Thus, CalO5 likely plays an identical role inthe biosynthesis of the calicheamicin aryltet-rasaccharide. In contrast, CalE8 demonstratesthe greatest sequence homology to two iter-ative PKSs involved in the biosynthesis of thepolyunsaturated fatty acids (PUFAs) eicosa-pentaenoic acid (EPA) in Shewanella (15,17) and docosahexaenoic acid (DHA) inMoritella marina (16, 17).

To confirm that CalE8 is critical to thebiosynthesis of calicheamicin in Micromonos-pora, a calE8 gene disruption was constructedby introduction of an apramycin selectablemarker (Fig. 3A). The calicheamicin producerM. echinospora is resistant to most antibiotics,and is inherently indisposed to genetic manip-ulation, with a best reported transformation ef-ficiency of 10!7 transformants per microgramDNA (18). In spite of these difficulties, a com-bination of many gene disruption attempts in M.echinospora LL6000 (19) led to nine indepen-dent apramycin-resistant clones. All nine iso-lates mapped consistently with the expectedcalE8 gene disruption by both PCR fragmentamplification and Southern hybridization. Therepresentative hybridization results from twoof these clones (pJAW117/LL6000-1 andpJAW117/LL6000-3) are illustrated in Fig.3B. All nine calE8 disruption mutants andtwo representative LL6000 isolates were sub-

Fig. 1. The structure of the nonchromoprotein enediyne calicheamicin. The enediyne families aretypically distinguished by either the number of carbons in the enediyne ring (9-membered versus10-membered) or by their association, or lack thereof, with an apoprotein (chromoprotein versusnonchromoprotein). With one exception (N1999A), the 9-membered enediynes are chromoproteinenediynes, whereas all 10-membered enediynes to date are nonchromoprotein. The colors correlatethe calicheamicin final structure to the functions of genes within the calicheamicin locus (Fig. 2)with the specific PKS structural components designated as PKS E and PKS O.

Fig. 2. The calicheami-cin locus from Mi-cromonospora echinos-pora spp. calichensis.The colors of genes de-lineate putative roles incalicheamicin biosyn-thesis, based on BLASTanalysis and correlateby color to thecalicheamicin structurepresented in Fig. 1. Thecorresponding calE8translation, CalE8, isalso shown to highlightthe location of themaindomains KS, AT, KR, DH,and TD.

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Ahlert, et al. (2002) Science 297, 1173

CalS13 TDP-4-keto-6-deoxyglucose aminotransferase (PDB 4ZAS) Wang, et al. 2015 ACS Chem. Biol.

CalG3

CalS8 + CalS13

CalS11

CalG2 CalG1 CalG4

SgcC5 type II peptide synthetase (PDB 4ZXW; Michalska et al. 2015)

Lohman, et al. (2013) Biochemistry 53, 902

LnmA with TG25 LnmZ with TG25D3 Two homologous P450 monooxygenases modify different sites of LNM, supported by docking models (to publish)

Polyketide synthases for antitumor antibiotic biosynthesis

ozmQ β-ketoacyl synthase (PDB 4OQJ; Nocek, et al. 2014)

Oxazolomycin (Streptomyces albus)

ozmH β-ketoacyl synthase domains (4221-4804) & (6118-6552) (PDB 4OPE, Osipuik, et al. 2014; 4OPF, Osipiuk, et al. 2014)

Oxazolomycin (OZM), a hybrid peptide-polyketide antibiotic, exhibits potent antitumor and antiviral activities. The methoxymalonate-derived moiety is highlighted (box). OZM biosynthesis has the following novel features: (i) hybrid nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) biosynthetic machinery that initiates peptide-polyketide biosynthesis with an oxazole moiety, elongates the peptide-polyketide chain by transitioning between NRPS-PKS four times, and terminates the peptide-polyketide synthesis with a β-lactone/ ϒ-lactam structure; (ii) a modular PKS that introduces all five of the C-methyl groups by choosing malonyl coenzyme A (CoA) as a chain extender followed by C methylation instead of using methylmalonyl CoA; (iii) specific control of double-bond geometry by the modular PKS to create the unique (Z, Z, E)- triene and (E, E)-diene; and (iv) introduction of the C16 and C13’ carbons, which is expected to require novel chemistry by the PKS or NRPS. (Zhao, et al. (2006) J. Bacteriol. 188, 4142)

Cloning of the methoxymalonyl-ACP biosynthesis locus fromS. albus JA3453. Degenerate primers (HADH-FP [5!-GTC CTGGGC GCC GGS GTS ATG GG-3!] and HADH-RP [5!-GTTGTC CAG GCC GAT SAG GTC NGC-3!]) were designed ac-cording to two conserved regions (VLGAGVMG and ADLIGLDN) of 3-hydroxyacyl-CoA dehydrogenases (HADHs) knownfor methoxymalonyl-ACP biosynthesis (9, 10, 20, 27–29) and usedto amplify putative HADH genes by PCR from S. albus JA3453total DNA using conditions suitable for the GC-rich DNA (8). Adistinct product with the predicted size of 0.73 kb was amplified,cloned into pGEM-EZ (Promega, Madison, WI) as pJTU1051,and sequenced. The deduced 228 amino acids showed high se-quence homology to Asm13 (59% identity), a known HADHcharacterized from the ansamitocin biosynthesis gene cluster (28).This HADH fragment was then used as a probe to screen the S.albus JA3453 genomic library prepared in pOJ446 (8) by standardmethods (8, 21). Of the 3,000 colonies screened by colony hybrid-ization, four overlapping cosmids, as represented by pJTU1059and pJTU1060, were identified and confirmed by PCR andSouthern hybridization to contain the 4.5-kb BglII fragment thatincludes the HADH probe (Fig. 2).

Genes encoding acyl-CoA dehydrogenases (ADHs) have beenclustered, without exception, with those encoding HADHs withinall methoxymalonyl-ACP biosynthesis loci characterized to date.Degenerate primers (ADH-FP [5!-CAG GGC ATG GCCGCS TGG ACS GT-3!] and ADH-RP [5!-GCA GGC GCGCAG GAT SCC SAC RCA-3!]) were similarly designed accord-ing to two conserved regions (QGMVVWTV and CVGILRAC)of ADHs from known methoxymalonyl-ACP biosynthesis loci (9,10, 20, 27–29) and used to examine the four HADH-positivecosmids by PCR for the presence of an ADH gene. A distinctproduct with the predicted size of 0.5 kb was readily amplifiedfrom pJTU1059 and cloned (pJTU1052), whose identity as anADH gene was confirmed by DNA sequencing.

Confirmation of the cloned HADH/ADH locus is essentialfor OZM biosynthesis. To confirm that the cloned locus en-codes OZM biosynthesis genes, a 12-kb BglII fragment inter-nal to pJTU1059 was replaced with the 1.1-kb thiostrepton (tsr)resistance (Thir) gene, suitable for selection in Streptomyces, toyield pJTU1064 (Fig. 3A). Introduction of pJTU1064 into S.albus JA3453 by conjugation with Thir selection, using pub-lished protocols (8, 21) with genetic methods we developed forJA3453 (unpublished work), yielded exconjugants at a fre-quency of about 10"5 exconjugants per donor. Since pJTU1064contains the SCP2* origin of replication that is functional butunstable in most Streptomyces strains (8), exconjugates that werealso apramycin resistant (Aprr) most likely had pJTU1064 inte-grated into the S. albus JA3453 chromosome via a single-cross-over homologous recombination event. Such Thir and Aprr col-onies were further screened on mannitol soy flour (MS) medium(8) for the Thir and Aprs phenotype. Approximately 1% of thecolonies examined were Aprs, due to loss of the SCP2*-derivedvector part of pJTU1064 as the result of the second crossoverhomologous recombination event (Fig. 3A). This genotype wasconfirmed for two of the Thir and Aprs isolates by Southernanalysis, in which a distinctive band at 12.2 kb in the wild-typeJA3453 strain was shifted to 1.5 kb in the ZH4 mutant strains(Fig. 3B). The ZH4 mutant strain was subsequently fermented totest for OZM production with the JA3453 wild-type strain as apositive control under identical conditions. High-performance liq-

FIG. 1. (A) Structures of oxazolomycin and related natural prod-ucts and (B) selected examples of natural products that utilize methoxy-malonate as a biosynthetic precursor. The methoxymalonate-derivedmoiety is highlighted (box). TTM, tautomycin.

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uid chromatography (HPLC) analysis showed a complete abol-ishment of OZM production in ZH4 (Fig. 3C), confirming theessential role the cloned locus plays in OZM biosynthesis.

Sequence analysis of the methoxymalonyl-ACP biosynthesislocus and localization of the complete ozm biosynthetic genecluster. The 12.2-kb PvuII fragment containing the PCR-am-plified HADH and ADH fragments was subcloned (pJTU1078).Sequencing of this region (12,249 bp) revealed 10 open readingframes (ORFS) [orf(-1) to (-3) and ozmA to -G] and an incom-plete ORF, ozmH (Fig. 2B). The ozmBCDEFG genes are co-transcribed in the same orientation, presumably as an operon,since the numbers of the nucleotides between stop and startcodons of the adjacent genes (i.e., 1 bp overlapping forozmG and ozmF; 14 bp between ozmF and ozmE; 16 bpbetween ozmE and ozmD; 4 bp overlapping for ozmD andozmC; 16 bp between ozmC and ozmB) are all too small tocode any regulatory element for transcriptional initiation.

Five of the six genes within this apparent operon (with theexception of ozmC) are absolutely conserved among methoxy-malonyl-ACP biosynthetic loci known to date. For example,OzmG (287 amino acids) showed significant homology tomembers of the HADH family of enzymes, such as Asm13(61% identity) (28), GdmK (61% identity) (20), FkbK (62%identity) (27), SorD (40% identity) (10, 29), and TtmE (64%identity) (9). Similarly, OzmF (221 amino acids) is a probableO-methyltransferase, OzmE is a probable ACP, OzmD is anADH, and OzmB is an activating enzyme to channel a glyco-lytic pathway intermediate to methoxymalonyl-ACP biosynthe-sis, all of which have homologs in known methoxymalonyl-ACPbiosynthetic loci (9, 10, 20, 27–29). Unique to the ozm locus isthe presence of ozmC, whose deduced product showed signif-icant sequence homology to DpsC (26% identity) from Strep-tomyces peucetius for doxorubicin biosynthesis. DpsC has beenproposed to play a role in selecting the methylmalonyl-CoAstarter unit for the Dps PKS (1). A similar function could beenvisaged for OzmC to activate the glycolytic pathway inter-mediate as glyceryl-ACP to initiate methoxymalonyl-ACP bio-

synthesis, a function that currently has been assigned solely toOzmB and its homologs (9, 10, 20, 27–29).

Upstream of ozmB is a divergently transcribed gene, ozmA,whose deduced product resembles a family of multidrug trans-porters, such as SgcB (28% identity) in C-1027 biosynthesisfrom Streptomyces globisporus (11), RemN (26% identity), in-volved in resistomycin biosynthesis, from Streptomyces resist-omycificus (6), and EncT (27% identity) in enterocin biosyn-thesis from Streptomyces maritimus (19). Therefore, OzmAcould act as an OZM-specific transporter to confer OZM resis-tance in S. albus JA3453. The three additional ORFs, orf(-3),orf(-2), and orf(-1), at the upstream location of the sequencedregion encode proteins whose functions were not apparent inOZM biosynthesis and hence may represent one boundary of theozm cluster (Fig. 2B).

The deduced product (397 amino acids) of the partial ORF,ozmH, downstream of the sequence region showed high se-quence homology to AT-less PKSs that are known for hybridpeptide-polyketide biosynthesis, such as LnmI (59% identity),involved in leinamycin biosynthesis, from Streptomyces atrooli-vaceus (25), and PedF (52% identity), involved in pederinbiosynthesis, from Paederus fuscipes (18). Colocalization ofgenes encoding hybrid NRPS-PKS with the methoxymalonyl-ACP biosynthesis locus within the ozm biosynthetic gene clus-ter is consistent with the hybrid peptide-polyketide biosyn-thetic origin of OZM (2). Sequential chromosomal walkingfrom the confirmed methoxymalonyl-ACP biosynthetic locusled to the eventual localization of the complete ozm clusterwithin a 135-kb DNA region as represented by the overlappingcosmids of pJTU1059, pJTU1060, pJTU1061, pJTU1062, andpJTU1063, the downstream boundary of which has been pre-liminarily determined by shotgun sequencing (Fig. 2A).

ozmC inactivation and complementation. To provide directevidence that ozmC is required for OZM biosynthesis, it wasinactivated by the PCR targeting and !-RED-mediated genereplacement method (3) by means of an in-frame deletionstrategy to eliminate any possible polar effect on the other

FIG. 2. (A) A 135-kb contiguous DNA from S. albus JA3453 covering the entire ozm gene cluster as represented by five overlapping cosmidclones. Probes used to clone and map the ozm gene cluster are designated Probe-1, -2, -3, and -4. (B) Genetic organization of the ozmmethoxymalonyl-ACP biosynthetic locus identified within the sequenced 12.2-kb PvuII fragment. B, BglII; P, PvuII.

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C-1027 is a potent antitumor agent with a previously undescribed molecular architecture and mode of action. Cloning and characterization of the 85-kilobase C-1027 biosynthesis gene cluster from Streptomyces globisporus revealed (i) an iterative type I polyketide synthase (ii) a general polyketide pathway for the biosynthesis of both the 9- and 10-membered enediyne antibiotics, and (iii) a convergent biosynthetic strategy for the C-1027 chromophore from four building blocks. Manipulation of genes governing C-1027 biosynthesis allowed us to produce an enediyne compound in a predicted manner to reduce toxicity for application in chemotherapy as enediyne-antibody conjugates

Calicheamicin γ1 is the most prominent nonchromoprotein enediyne, as an antitumor agent (> 5000 times than adriamycin) with two distinct structural regions: The aryltetrasaccharide is composed of a set of carbohydrate and aromatic units, which confers specificity to the minor groove of DNA; the aglycone consists of a highly functionalized bicyclo[7.3.1]tridecadiynene core structure with an allylic trisulfide for the initial trigger for cycloaromatization. Once the aryltetrasaccharide is docked to DNA, aromatization of the bicyclo[7.3.1]tridecadiynene core structure, via a 1,4-dehydrobenzene-diradical, results in the site-specific oxidative double-strand scission of the targeted DNA

been identified from recent genome sequencing efforts, all ofwhich, however, were annotated as proteins of unknownfunction (Figure S1 of the Supporting Information).Previously, we have demonstrated that the β-branched C3

unit in LNM biosynthesis is installed by a set of four proteins,LnmL, an ACP, LnmK, a bifunctional AT/DC, LnmM, a HCS,and LnmF, an ECH1.8,9 Inactivation of lnmK, lnmL, or lnmM inS. atroolivaceus S-140 abolished LNM production; the resultantmutant strains instead accumulated an identical set of shuntmetabolites, all of which lack the β-branched C3 unit.9 In vitrobiochemical investigation confirmed that LnmK is a bifunc-tional AT/DC: it is first self-acylated using methylmalonyl-CoA, the methylmalonyl group is then transferred from LnmKto the phosphopantetheinyl group of LnmL, and finally theresultant methylmalonyl-S-LnmL is decarboxylated to affordpropionyl-S-LnmL (Figure 1B).8 LnmM-catalyzed condensa-tion between propionyl-S-LnmL and the β-keto group of thegrowing polyketide intermediate, tethered to the ACP domainof LnmJ PKS module 8, followed by LnmF-catalyzeddehydration, completes the installation of the β-branch C3unit into LNM (Figure 1A).9 While homologues of LnmF,LnmL, and LnmM are known, and their functions have beenwell characterized from several biosynthetic pathways of β-branched polyketides,10−16 LnmK shows no sequence homol-ogy to proteins of known function, representing a new family ofAT/DC enzymes.8,9

Here we report the X-ray structure of the bifunctional AT/DC enzyme LnmK. LnmK is a homodimer with each of themonomers adopting a double-hot-dog fold (DHDF). Cocrys-

tallization of LnmK with methylmalonyl-CoA revealed an activesite tunnel terminated by residues from the dimer partner. Incontrast to canonical AT and KS enzymes that employ Ser orCys as an active site residue, none of these residues are found inthe vicinity of the LnmK active site. Instead, three tyrosineswere identified, one of which, Tyr62, was established, by site-directed mutagenesis, to be the most likely active site residuefor the AT activity of LnmK. LnmK represents the first ATenzyme that employs a Tyr as an active site residue and the firstidentified member of the family of DHDF enzymes thatdisplays an AT activity. The LnmK structure sets the stage forprobing the DC activity of LnmK through site-directedmutagenesis. These findings highlight natural product bio-synthetic machinery as a rich source of novel enzyme activities,mechanisms, and structures.

■ MATERIALS AND METHODSChemicals and Reagents. DL-2-[methyl-14C]Malonyl-CoA

(American Radiolabeled Chemicals, St. Louis, MO), DL-methylmalonyl-CoA lithium salt (Sigma-Aldrich, St. Louis,MO), dNTPs (New England Biolabs, Ipswich, MA), and allother common biochemicals and chemicals were from standardcommercial sources. Restriction enzymes and T4 DNA ligase(New England Biolabs), T4 DNA polymerase (Lucigen,Middleton, WI), and Platinum Pfx DNA polymerase(Invitrogen, Carlsbad, CA) were purchased. The pRSFDuet-1plasmid and Escherichia coli NovaBlue and BL21(DE3) cellswere from Novagen (Madison, WI). Primer synthesis and DNA

Figure 1. (A) Proposed LNM biosynthetic pathway featuring LnmKLM-catalyzed β-alkylation of the LnmJ PKS module 8 ACP-tetheredintermediate in S. atroolivaceus S-140. (B) Two parallel but distinct pathways for the acyl-S-ACP substrate involved in β-alkylation in polyketidebiosynthesis, i.e., acetyl-S-ACP from malonyl-CoA by dedicated ACP, AT, and KS enzymes and propionyl-S-ACP from methylmalonyl-CoA by adedicated ACP and a bifunctional AT/DC enzyme.

Biochemistry Article

dx.doi.org/10.1021/bi301652y | Biochemistry 2013, 52, 902−911903

LnmK (PDB 4NNQ) bifunctional acyltransferase/decarboxylase (Lohman)

LnmF putative enoyl-CoA hydratase (PDB 4NNQ; Michalska et al. 2014)

NN

S

S S

O

O

O

O

OH

H

OH

8

2'

LnmA LnmZ

Leinamycin

Leinamycin (LNM), a potent antitumor antibiotic (i) its backbone is assembled from the amino acid and short carboxylic acid precursors by a hybrid nonribosomal peptide synthetase (NRPS)-acyltransferase-less type I polyketide synthase (PKS)3−5 and (ii) the β-branched C3 unit, which is a part of the unique five-membered 1,3-dioxo-1,2-dithiolane moiety, is installed by a novel pathway for β-alkylation in polyketide biosynthesis Tang, et al. (2004) Chem. Biol. 1, 33

Conclusion Our collaborative multicenter high throughput pipeline from protein production to structure solving with complementary in vitro and in vivo approaches proved to be successful in the discovery of enzymes for natural product biosynthesis. Over 60 new structures covering >40 unique protein sequences from >10 species of Bacteria and Archaea were solved in the past 4 years. Most of these provide novel structural insights into the enzyme mechanisms and allow rational engineering of enzymes with novel functions or altered biosynthetic pathways for promising biomedical and industrial applications.

Migrastatin and iso-Migrastatin (Streptomyces platensis)

MgsF AT-less PKS domains (3137-3758) & (550-1188) (PDB 4TKT, Chang, et al. 2014; 4ZDN, Chang et al. 2014 )

KR and an enoylreductase (ER) domain in module-10 (Figures2 and S1).10a Additionally, four tailoring stepshydroxylationat C-8, O-methylation at HO-C-8, dehydration of the C-17 OHmoiety, and enoyl reduction of the C-16/C-17 olefinarerequired for converting 10 to 1; in fact, all possibleintermediates en route from 10 to 1 have been isolated fromwild-type S. platensis (Figures 2 and S1).1b However, based onbioinformatics, only three tailoring enzymes were identifiedwithin the mgs cluster, MgsI (an oxidoreductase), MgsJ (an O-methyltransferase), and MgsK (a P-450 hydroxylase), togetheraccounting for three of the four tailoring steps.10a While it hasbeen proposed that MgsI, MgsK, and MgsJ are responsible forenoyl reduction of the C-16/C-17 olefin, C-8 hydroxylation,and O-methylation of the HO-C-8, respectively, the exacttiming for each of the steps is unknown; also unclear is thenature of C-16/C-17 dehydration prior to enoyl reduction ofthe C-16/C-17 double bond by MgsI (Figures 2 and S1).10a

Here we report systematic inactivation of mgsIJK in S.platensis, and isolation and characterization of the resultingintermediates, with production time courses, from these mutantstrains. These studies enabled us to discover both 10 and 13 asthe nascent products of the iso-MGS AT-less type I PKS andaccount for the formation of all post-PKS biosynthetic

intermediates known to date (i.e., 10−17) by the threetailoring enzymes MgsIJK (Figure 2).First we systematically inactivated mgsI, mgsJ, and mgsK in

the S. platensis wild-type by replacing them individually or incombinations with an apramycin resistance gene cassette, usingthe λ-RED-mediated PCR-targeting mutagenesis strategies(Supporting Information).10a,12 The resultant mutant strainswere named SB11016 (i.e., ΔmgsI), SB11017 (i.e., ΔmgsJ),SB11018 (i.e., ΔmgsK), SB11019 (i.e., ΔmgsIJ), SB11020 (i.e.,ΔmgsJK), and SB11021 (i.e., ΔmgsIJK), whose genotypes wereconfirmed by Southern analysis (Figure S2).Next we fermented the S. platensis mutant strains, with the

wild-type as a control, to investigate the effect of thesemutations on 1 biosynthesis. Fermentation of the wild-type andmutant strains, isolation of 1 and intermediates 10−17, anddetermination of the metabolite profiles by HPLC analysisfollowed established procedures (Supporting Information).1,5,10

Authentic standards of 1 and 10−17 have been isolated fromthe S. platensis wild-type, and their structures wereunambiguously established by comprehensive MS and 1H and13C NMR analysis, with the exception of 11, which wasproduced in trace quantity by the wild-type.1b We re-isolated11 from SB11019 and unambiguously confirmed its structure

Figure 2. Proposed biosynthetic pathway for iso-MGS (1) featuring the iso-MGS AT-less type I PKS that lacks a DH domain in module-4, a MTdomain in module-5, a KR domain in module-8, and KR and ER domains in module-10 according to the collinear PKS model for the biosynthesis ofthe two nascent products, 10 and 13, and the tailoring enzymes MgsIJK with broad substrate specificity that convert 10 to 12 and 13 to 1.Biosynthesis of the glutarimide starter unit by the loading module, module-2, and module-3 has been previously proposed.10a Heavy arrows denotethe preferred pathway by the tailoring enzymes, with thin arrows accounting for metabolites resulted from substrate promiscuity. Abbreviations:ACP, acyl carrier protein; AMT, amidotransferase; DH, dehydratase; ER, enoylreductase; KR, ketoreductase; KS, ketosynthase; MT,methyltransferase; TE, thioesterase. [NH2] depicts the amino donor for the AMT domain, [SAM] denotes S-adenosylmethionine as the methyldonor for the MT domain, the ACP domain with an overlaid X is nonfunctional, the domain with ? denotes an unknown function, green ovals depictAT-docking domains, and the yellow domains highlight the missing domains for the iso-MGS AT-less type I PKS predicted according to thecollinear model for the biosynthesis of 10 and 13.10a,11

Journal of the American Chemical Society Communication

dx.doi.org/10.1021/ja4002635 | J. Am. Chem. Soc. 2013, 135, 2489−24922490

Post-Polyketide Synthase Steps in Iso-migrastatin Biosynthesis,Featuring Tailoring Enzymes with Broad Substrate SpecificityMing Ma,†,⊥ Thomas Kwong,†,⊥ Si-Kyu Lim,‡ Jianhua Ju,‡ Jeremy R. Lohman,† and Ben Shen*,†,‡,§,∥

†Department of Chemistry, §Department of Molecular Therapeutics, and ∥Natural Products Library Initiative, The Scripps ResearchInstitute, Jupiter, Florida 33458, United States‡Division of Pharmaceutical Sciences, School of Pharmacy, University of Wisconsin, Madison, Wisconsin 53705, United States

*S Supporting Information

ABSTRACT: The iso-migrastatin (iso-MGS) biosyntheticgene cluster from Streptomyces platensis NRRL 18993consists of 11 genes, featuring an acyltransferase (AT)-lesstype I polyketide synthase (PKS) and three tailoringenzymes MgsIJK. Systematic inactivation of mgsIJK in S.platensis enabled us to (i) identify two nascent products ofthe iso-MGS AT-less type I PKS, establishing anunprecedented novel feature for AT-less type I PKSs,and (ii) account for the formation of all known post-PKSbiosynthetic intermediates generated by the three tailoringenzymes MgsIJK, which possessed significant substratepromiscuities.

I so-migrastatin (iso-MGS, 1) belongs to the glutarimide-containing polyketide family of natural products; other

members of this family include migrastatin (MGS, 2),dorrigocin A (DGN A, 3), 13-epi-DGN A (4), DGN B (5),lactimidomycin (LTM, 6), cycloheximide (7), streptimidone(8), and 9-methylstreptimidone (9) (Figure 1).1 While 2 wasoriginally isolated from Streptomyces sp. MK929-43F1,2 and 3and 5 were isolated from Streptomyces platensis NRRL 18993,3

re-examination of the S. platensis fermentation revealed that thisstrain also produced 1 and 2.4 We subsequently established that1 was the only nascent natural product biosynthesized by S.platensis, and 2−5 resulted from H2O-mediated, non-enzymaticring-expansion and ring-opening rearrangements of 1 (Figure1).5

The glutarimide-containing polyketides exhibit a multitude ofbiological activities.1,6 As it was originally discovered, 2displayed moderate potency in cell migration inhibition assays,2

with synthetic mimics of the macrolide moiety displayingsignificantly improved potency.7 We previously generated afocused library of glutarimide-containing polyketides featuringthe molecular scaffolds 1−6, including eight biosyntheticcongeners of 1 (10−17, Figure 2) from optimizedfermentations of S. platensis1b and 6 and three of itsbiosynthetic congeners from Streptomyces amphibiosporusATCC 53964.5b,6a,b Preliminary screening of this libraryrevealed that 12-membered macrolides, as exemplified by 1and 6, were also potent cell migration inhibitors.6b The modesof action that dictate and differentiate cell migration inhibitionfrom cytotoxicity for the glutarimide-containing polyketidesremain controversial.1a While the actin-bundling protein fascinhas been identified as the target for the cell migration inhibitory

activity of 2,8 blocking the translocation step in eukaryoticprotein translation initiation has been deduced as themechanism for the cytotoxicity of 6.9

We previously cloned and sequenced the mgs biosyntheticgene cluster from S. platensis NRRL 18993, which consists of 11genes (mgsABCDEFGHIJK) (Figure S1).10 Inactivation ofselected genes in S. platensis10a and expression of the mgscluster in heterologous Streptomyces hosts10b−d unambiguouslyestablished that the 11 genes are necessary and sufficient toencode 1 production. The biosynthetic machinery of 1,featuring an acyltransferase (AT)-less type I polyketidesynthase (PKS), is characterized by several intriguing proper-ties.10a,11 On the assumption of 10 as the nascent PKS product,which has been isolated from the wild-type S. platensis,1b andthe collinear model for its biosynthesis,11 the iso-MGS AT-lesstype I PKS minimally lacks a methyltransferase (MT) domainin module-5, a ketoreductase (KR) domain in module-8, and a

Received: January 9, 2013Published: February 7, 2013

Figure 1. Structures of selected glutarimide-containing polyketidenatural products 1−9 and H2O-mediated, non-enzymatic ring-expansion and ring-opening rearrangements of 1 to 2−5.

Communication

pubs.acs.org/JACS

© 2013 American Chemical Society 2489 dx.doi.org/10.1021/ja4002635 | J. Am. Chem. Soc. 2013, 135, 2489−2492

Migrastatin (MGS) is a non-enzymatic product from iso-Migrastatin (iso-MGS); the latter is nascent biosynthetic product of mgsABCDEFGHIJK cluster. Both belong to glutarimide-containing polyketide family of natural products with varied inhibitory potency against tumor cell migration. (Ming, et al. (2013) JACS. 135, 2489)

of sMAT in the presence of ADP and methionine at65 °C for 90 min under standard assay conditions ledto < 2% AdoMet formation. Thus, two explanationsfor this unusual product formation have been pro-posed: (a) the ADP stock solution is contaminated bya sufficient amount of ATP; (b) the unusual reactioncatalyzed by sMAT can actually occur in vitro, butmay take as long as 1 month to complete, which cor-responds to the time of crystal growth in this experi-ment.

Structure homology

A DALI search [26] for structures similar to the sMATmonomer returned several hits, all of which are previ-ously solved MAT structures with Z-scores between 23and 29. These MAT structures share a very high level

of overall sequence identity (> 50%) and a high levelof conservation among residues associated with sub-strate binding. Interestingly, sMAT only has a maxi-mum sequence identity of 19% with these knownMATs, but shares a similar three-domain (Fig. 6A).

For the comparison of active site residues, crystalstructures of sMAT, eMAT [3] and hMAT2A [8] werealigned by ligands as described in Experimental proce-dures (Table 4). Surprisingly, 16 of 17 active site resi-dues detected in sMAT have an identical or similarresidue in eMAT and hMAT2A. The only extra resi-due sMAT has is H315, which forms a hydrogen bondwith O5 in diphosphate. DALI-based sequence alignmentwas able to identify 11 pairs of residues (Fig. 2). Eightof them are conserved among sMAT and other MATs,including the crucial residues histidine and lysine forthe proposed SN2-like mechanism [3]. The other threepairs are very similar residues at the same spot: forexample, in sMAT tyrosine 270 forms stacking interac-tions with the adenine ring of AdoEth/AdoMet, whilein eMAT it is phenylalanine 230. Intriguingly, thereare another five pairs of residues that are not detect-able via DALI search: the side chain of sMAT lysine 25(eMAT lysine 245) helps stabilize the triphosphategroup; the side chains of sMAT histidine 58 andasparagine 60 (eMAT glutamine 98 and lysine 269)form hydrogen bonds with the carboxyl group ofmethionine or ethionine; the side chain of sMAT glu-tamate 305 (eMAT aspartate 271) forms ionic bondswith the magnesium ion; the side chain of sMATlysine 63 occupies the same spot as the eMAT potas-sium ion and helps stabilize the diphosphate ligand(Fig. 6B).

Unlike other known MATs, it has been previouslyreported that the activity of sMAT cannot beenhanced by K+ [16]. In the present study, all thecrystals of sMAT were obtained from the crystalliza-tion condition containing more than 150 mM potas-sium, but electron density suitable for K+ was not

Table 4. Ligand-based alignment of active site residues between

sMAT and eMAT. Residues for proposed SN2 reaction are highlighted

in bold. The atom numbers used for interaction analysis here are the

numbers from the AdoEth bound structure (PDB code 4L2Z).

Interaction partner sMAT eMAT DALI alignment Chain

O1, O2 in PPi, O2 in PO4 K25 K245 Not detected B

O3 in PPi H29 H14 Detected B

Mg2+ D31 D16 Detected B

O7, O8 in AdoEth H58 Q98 Not detected A

O7, O8 in AdoEth N60 K269 Not detected A

K63 (K+ in eMAT) D62 E42 Detected A

O5, O7 in PPi K63 K+ – A

Ethyl group in AdoEth L145 I102 Detected A

O26 in AdoEth D199 D163 Detected B

O3 in PPi K201 K165 Detected B

Stacked with adenine ring Y270 F230 Detected B

O27 in AdoEth D282 D238 Detected B

O1, O3 in PO4 R288 R244 Detected B

Mg2+ E305 D271 Not detected A

O6 in PPi, O1 in PO4 K310 K265 Detected A

O5 in PPi H315 – – A

Ethyl group in AdoEth I349 I302 Detected A

AdoEth

ADP

Chemicals added in co-crystallization buffer Chemicals built in the final model

Ethionine

PPi

PO43–PO43–

Mg2+

Mg2+

Mg2+

Mg2+

Fig. 5. The active site of sMAT with the

simulated annealing Fo ! Fc omit map

(contoured at 3.0r). Substrates including

ADP, ethionine, PO43! and Mg2+ are

modeled on the left. Products including

AdoEth, PPi, PO43! and Mg2+ are

modeled on the right.

4234 FEBS Journal 281 (2014) 4224–4239 ª 2014 FEBS

Crystal structure of sMAT F. Wang et al.

observed in any data sets. In addition, potassiumdependence has been previously reported in a closeMAT homolog from Methanococcus jannaschii, whichshares the same active site residues with sMAT exceptfor the lysine [17]. Combined with the active sitesalignment evidence above (Fig. 6B), it is very likelythat the catalytic activity of sMAT is not affected byK+, because the lysine in sMAT serves to present therequisite cation properties.

Interestingly, eMAT and hMAT2A also have someability to incorporate ethionine. The ethionine turnsover with sMAT and hMAT2A is near 100% whereaswith eMAT is just 10% [15]. A ligand-based alignment(Fig. 6C) shows that sMAT has a larger cavity aroundthe ethyl/methyl group than either hMAT2A oreMAT. Placement of the ethyl group in eMAT willcause serious clashes with isoleucine 102 and 302,while in hMAT2A the ethionine causes moderateclashes with isoleucine 139 and 344. In sMAT a leu-cine (L145) is substituted for one of the conserved iso-

leucines in other MATs (isoleucine 102 in eMAT),which provides more active site flexibility for ethylgroup binding. The ethyl group in sMAT only hasminor clashes with leucine 145 and isoleucine 349.Therefore, it is very likely that the better proficiency ofsMAT is caused by a larger cavity adjacent to themethyl/ethyl group. Also, branched analogs high-lighted in Fig. 1B turn over significantly better withsMAT, comparing with eMAT, hMAT2A andmjMAT [15]. Interestingly, mjMAT is a thermophilicarchaeal MAT that has all active site residues con-served with sMAT. However, further comparisonbetween their active site cavities cannot be conductedbecause mjMAT structure remains unknown. The cur-rent structural information suggests that the betterturnover rate of branched AdoMet analogs withsMAT is possibly mediated by some general orienta-tion/dynamics of the gating loop and/or secondaryshell variations. The specific residues contributing tothis are currently unknown.

B

C

eMATsMAT Alignment

K63 K+

AdoEth

L145

AdoMet

sMAT eMAThMAT2A

I349 N159

D160 D156 D118

G155 G117I344

I139 I102

I302

3.5

3.6

3.8

2.9

3 .2

3 .2

3.1 3.1

3 .4

2.9

2.9 3.2

3 .0

ethyl group ethyl group ethyl group

A

sMAT eMAThMAT2A

Fig. 6. Folding and active site

comparisons between sMAT, hMAT2A

and eMAT. (A) Monomers of sMAT,

hMAT2A and eMAT show a similar three-

domain fold. (B) Substrate-based

alignment of sMAT and eMAT shows a

space overlap between lysine in sMAT

and K+ in eMAT. (C) Substrate-based

alignment of sMAT, hMAT2A and eMAT

displays the clashes around the sMAT

ethyl group. The clashes are calculated by

‘show bumps’ in PYMOL and the serious/

medium/minor clashes are shown in red/

brown/green.

FEBS Journal 281 (2014) 4224–4239 ª 2014 FEBS 4235

F. Wang et al. Crystal structure of sMAT

Simulated annealing Fo-Fc omit map at 3σ

Wang, et al. (2014) FEBS J. 281, 4224 SAM synthetase with AdoEth (PDB 4L2Z)

the adenine ring also form a hydrogen bond networkto the enzyme. The 20-OH and 30-OH of the adeno-sine ribose interact with the side chains of aspartate199, aspartate 282 and serine 277. Similar interactionsinvolving aspartic acids have been seen in other MATstructures bound with AdoMet, but not for serine[3,8]. The methionine/ethionine moiety (of AdoMetand AdoEth, respectively) forms hydrogen bonds withfour residues, in which the amino group interactswith the side chain of aspartate 282 and the carboxyl-ate group interacts with the side chain of histidine 58,

asparagine 60 and asparagine 159. The methyl orethyl group is buried in a slightly hydrophobic pocketsurrounded by asparagine 159, aspartate 160, isoleu-cine 349, leucine 145 and the adenine ring. Like simi-lar observations in eMAT [3], the PPi and Pi form aU-shaped conformation with two magnesium ions clo-sely stacked on both sides. Further, the two magne-sium sites are formed with the side chains ofaspartate 31, glutamate 305 and three water mole-cules. The phosphate groups are surrounded and sta-bilized by the side chains of several basic amino

A

90o

C D

apo sMATAdoEth-bound sMAT

Gating loop

Gating loop Gating loop

Gating loop

Subunit A Subunit B

Subunit C Subunit D

Gating loop

B

Fig. 3. The overall molecular structure and

active site contents of sMAT. (A) Tetramer

assembly of sMAT in the crystal structure

as calculated by PISA. The surface of the

protein is displayed: four protein

monomers are shown in light blue or

yellow and the gating loops are shown in

red. One protein monomer is displayed on

the right showing the three intertwined

domains as cartoon and the ligands as

sticks. (B) Stereoview of sMAT–ligand

interactions. The stick model of AdoEth,

PPi, PO43! and Mg2+ is depicted in

spheres and the interacting sMAT

residues are labeled and illustrated in

green. (C) Side view of sMAT dimer with

AdoEth bound. The gating loop region is

highlighted in red. (D) Side view of apo

sMAT dimer. The gating loop region is

highlighted in red.

FEBS Journal 281 (2014) 4224–4239 ª 2014 FEBS 4229

F. Wang et al. Crystal structure of sMAT

Sulfolobus solfataricus SAM synthetase shows slightly enhanced substrate promiscuity toward various unnatural substrate than reported MAT from other organisms due to active site cavity shape/size variation. sMAT active site rational variants enhance unnatural product synthesis.(unpublished work, Wang F. et al).

WecE (E. coli) TDP-4-keto-6-deoxyglucose aminotransferase (PDB 4ZAS) Wang, et al. 2015 ACS Chem. Biol.

AtmS13 (Actinomadura melliaura) TDP-4-keto-2,6-dideoxy-α-D-xylose aminotransferase (PDB 4ZWV) Shanteri, et al. 2015 Proteins

Plu4264 cupin (PDB 4Q29) (Photorhabdus luminescens) Weerth, et al. 2015 Proteins 83,383

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ZbmA (PDB 4IAG) zorbamycin binding (Streptomyces flavoviridis) Cuff, et al. 2013

Blml type II ACP (PDB 4NEO) (Streptomyces verticillus) Lohman, et al. 2014 Proteins 82,1210

TlmII (Streptoalloteichus hindustanus) tallysomycin condensation protein 4HVM Chang, et al 2014

SsfS6 (Streptomyces sp.) tetracycline polyketide C-glycosyltransferase 4G2T Wang, et al. 2013 Proteins 81

galactose mutarotase (PDB 4RNL) Tan, et al 2014 (Streptomyces platensis )

PriB (Streptomyces sp.) Indole Prenyltransferase Cao H, et al (unpublished)

MgsE AT-less PKS domain (2542-3153) (PDB 4QYR; Kim, et al. 2014)