Unique features of the ketosynthase domain in a nonribosomal peptide synthetase–polyketide synthase hybrid enzyme, tenuazonic acid synthetase 1

Abstract

Many microbial secondary metabolites are produced by multienzyme complexes comprising nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs). The ketosynthase (KS) domains of polyketide synthase normally catalyze the decarboxylative Claisen condensation of acyl and malonyl blocks to extend the polyketide chain. However, the terminal KS domain in tenuazonic acid synthetase 1 (TAS1) from the fungus Pyricularia oryzae conducts substrate cyclization. Here, we report on the unique features of the KS domain in TAS1. We observed that this domain is monomeric, not dimeric as is typical for KSs. Analysis of a 1.68-Å resolution crystal structure suggests that the substrate cyclization is triggered via proton abstraction from the active methylene moiety in the substrate by a catalytic His-322 residue. Additionally, we show that TAS1 KS promiscuously accepts aminoacyl substrates and that this promiscuity can be increased by a single amino acid substitution in the substrate-binding pocket of the enzyme. These findings provide insight into a KS domain that accepts the amino acid–containing substrate in an NRPS–PKS hybrid enzyme and provide hints to the substrate cyclization mechanism performed by the KS domain in the biosynthesis of the mycotoxin tenuazonic acid. Many microbial secondary metabolites are produced by multienzyme complexes comprising nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs). The ketosynthase (KS) domains of polyketide synthase normally catalyze the decarboxylative Claisen condensation of acyl and malonyl blocks to extend the polyketide chain. However, the terminal KS domain in tenuazonic acid synthetase 1 (TAS1) from the fungus Pyricularia oryzae conducts substrate cyclization. Here, we report on the unique features of the KS domain in TAS1. We observed that this domain is monomeric, not dimeric as is typical for KSs. Analysis of a 1.68-Å resolution crystal structure suggests that the substrate cyclization is triggered via proton abstraction from the active methylene moiety in the substrate by a catalytic His-322 residue. Additionally, we show that TAS1 KS promiscuously accepts aminoacyl substrates and that this promiscuity can be increased by a single amino acid substitution in the substrate-binding pocket of the enzyme. These findings provide insight into a KS domain that accepts the amino acid–containing substrate in an NRPS–PKS hybrid enzyme and provide hints to the substrate cyclization mechanism performed by the KS domain in the biosynthesis of the mycotoxin tenuazonic acid. We previously reported that tetramic acid (2,4-pyrrolidinedione) derivative mycotoxin tenuazonic acid (TeA) is synthesized by tenuazonic acid synthetase 1 (TAS1) in Pyricularia oryzae. TAS1 is the first reported fungal nonribosomal peptide synthetase–polyketide synthase (NRPS–PKS) hybrid enzyme that consists of an NRPS module of domains C-A-PCP (C, condensation; A, adenylation; PCP, peptidyl carrier protein) and a terminal PKS ketosynthase (KS) domain. Tetramic acid ring formation by Dieckmann cyclization in the course of TeA biosynthesis is predicted to be catalyzed by the KS domain (1Yun C.-S. Motoyama T. Osada H. Biosynthesis of the mycotoxin tenuazonic acid by a fungal NRPS-PKS hybrid enzyme.Nat. Commun. 2015; 6 (26503170): 875810.1038/ncomms9758Crossref PubMed Scopus (79) Google Scholar) (Fig. 1A). The KS domain is an essential domain of modular type I PKS systems that normally carries out decarboxylative Claisen condensation of acyl and malonyl blocks to yield linear polyketide backbones (2Hertweck C. The biosynthetic logic of polyketide diversity.Angew. Chem. Int. Ed. 2009; 48 (19514004): 4688-471610.1002/anie.200806121Crossref PubMed Scopus (891) Google Scholar) (Fig. 1B). However, exceptional KS domains have also been reported to have a noncanonical role in type I PKS systems. PKS RhiE-KS3 plays a key role in vinylogous chain branching in the course of rhizoxin biosynthesis without ketide chain elongation (3Bretschneider T. Heim J.B. Heine D. Winkler R. Busch B. Kusebauch B. Stehle T. Zocher G. Hertweck C. Vinylogous chain branching catalysed by a dedicated polyketide synthase module.Nature. 2013; 502 (24048471): 124-12810.1038/nature12588Crossref PubMed Scopus (83) Google Scholar). A KS domain which has one His residue of the catalytic triad mutated has been reported to only carry out substrate transfer to the next domain in the course of FR901464 biosynthesis (4He H.-Y. Tang M.-C. Zhang F. Tang G.-L. Cis-double bond formation by thioesterase and transfer by ketosynthase in FR901464 biosynthesis.J. Am. Chem. Soc. 2014; 136 (24617828): 4488-449110.1021/ja500942yCrossref PubMed Scopus (30) Google Scholar). Additionally, in type III PKSs homodimers of KS domains catalyze ketide extension and substrate cyclization in a single catalytic pocket (5Katsuyama Y. Ohnishi Y. Type III polyketide synthases in microorganisms.Methods Enzymol. 2012; 515 (22999182): 359-37710.1016/B978-0-12-394290-6.00017-3Crossref PubMed Scopus (33) Google Scholar). Nevertheless, a KS domain with a cyclization and not ketide extension role has only been reported in TAS1. NRPSs have a monomeric structure (6Tanovic A. Samel S.A. Essen L.-O. Marahiel M.A. Crystal structure of the termination module of a nonribosomal peptide synthetase.Science. 2008; 321 (18583577): 659-66310.1126/science.1159850Crossref PubMed Scopus (246) Google Scholar), but the EM reconstruction of full-length type I PKS module shows an overall homodimeric architecture within which the KS domains associate (7Dutta S. Whicher J.R. Hansen D.A. Hale W.A. Chemler J.A. Congdon G.R. Narayan A.R.H. Håkansson K. Sherman D.H. Smith J.L. Skiniotis G. Structure of a modular polyketide synthase.Nature. 2014; 510 (24965652): 512-51710.1038/nature13423Crossref PubMed Scopus (204) Google Scholar) and KS domain dimerization is a common feature in all reported PKS structure (8Weissman K.J. Uncovering the structures of modular polyketide synthase.Nat. Prod. Rep. 2015; 32 (25310997): 436-45310.1039/c4np00098fCrossref PubMed Google Scholar). Additionally, there is a report showing PKSs to be dimers that rely on biochemistry, not structure (9Kao C. Pieper M.R. Cane D.E. Khosla C. Evidence for two catalytically independent clusters of active sites in a functional modular polyketide synthase.Biochemistry. 1996; 35 (8823171): 12363-1236810.1021/bi9616312Crossref PubMed Scopus (88) Google Scholar). However, there is no report on the association state of the amino acid–containing substrate-accepting KS domain in the NRPS–PKS hybrid enzyme like TAS1. Bioactive natural products, biosynthesized by the multidomain enzymes NRPS and PKS, often possess a cyclic portion (10Fischbach M.A. Walsh C.T. Assembly-line enzymology for polyketide and nonribosomal peptide antibiotics: Logic, machinery, and mechanisms.Chem. Rev. 2006; 106 (16895337): 3468-349610.1021/cr0503097Crossref PubMed Scopus (1077) Google Scholar, 11Walsh C.T. Polyketide and nonribosomal peptide antibiotics: Modularity and versatility.Science. 2004; 303 (15031493): 1805-181010.1126/science.1094318Crossref PubMed Scopus (476) Google Scholar). Because of the beneficial properties such as cell permeability and biological activity, cyclic natural products have attracted considerable attention in the field of therapeutic agent development (12Abdalla M.A. McGaw L.J. Natural cyclic peptides as an attractive modality for therapeutics: A mini review.Molecules. 2018; 23 (30127265): 208010.3390/molecules23082080Crossref Scopus (68) Google Scholar, 13Zorzi A. Deyle K. Heinis C. Cyclic peptide therapeutics: Past, present and future.Curr. Opin. Chem. Biol. 2017; 38 (28249193): 24-2910.1016/j.cbpa.2017.02.006Crossref PubMed Scopus (374) Google Scholar, 14Driggers E.M. Hale S.P. Lee J. Terrett N.K. The exploration of macrocycles for drug discovery—an underexploited structural class.Nat. Rev. Drug. Discov. 2008; 7 (18591981): 608-62410.1038/nrd2590Crossref PubMed Scopus (984) Google Scholar). Generally, bacterial NRPSs utilize the terminal thioesterase (TE) domain to conduct substrate cyclization, whereas fungal NRPSs use the terminal condensation-like domain for product cyclization. Additionally, the terminal reductase-like cyclization domain in fungal PKS–NRPS hybrid enzymes known that related to substrate cyclization (15Trauger J.W. Kohli R.M. Mootz H.D. Marahiel M.A. Walsh C.T. Peptide cyclization catalysed by the thioesterase domain of tyrocidine synthetase.Nature. 2000; 407 (11001063): 215-21810.1038/35025116Crossref PubMed Scopus (260) Google Scholar, 16Gao X. Haynes S.W. Ames B.D. Wang P. Vien L.P. Walsh C.T. Tang Y. Cyclization of fungal nonribosomal peptides by a terminal condensation-like domain.Nat. Chem. Biol. 2012; 8 (22902615): 823-83010.1038/nchembio.1047Crossref PubMed Scopus (121) Google Scholar, 17Boettger D. Hertweck C. Molecular diversity sculpted by fungal PKS-NRPS hybrids.Chembiochem. 2013; 14 (23225733): 28-4210.1002/cbic.201200624Crossref PubMed Scopus (149) Google Scholar) (Fig. 1C). However, the precise cyclization mechanism at the molecular level of all the reported cyclization domains remains unknown. Herein, we propose the mechanism of cyclization for tetramic acid ring formation by the KS domain of TAS1 in the course of TeA biosynthesis using crystallographic and mutational analyses and demonstrate broad substrate specificity and characteristic association state of TAS1 KS. We first wished to determine the oligomerization state of the TAS1 KS domain in solution. To obtain the excised KS domain, a gene construct encoding only the KS domain from TAS1 was heterologously expressed in Escherichia coli. This KS domain was purified, had its affinity tags removed, and was confirmed to be active (Fig. S1). Intriguingly, size-exclusion chromatography showed TAS1 KS domain to be in monomeric state in aqueous solution (Fig. 2). To determine whether this state is distinctive to TAS1 KS or common to amino acid–containing substrate-accepting KS, we also performed size-exclusion chromatography analysis of OzmQ-KS1 protein, which accepts aminoacyl substrate but preforms traditional elongation in the hybrid oxazolomycin biosynthetic gene cluster (18Zhao C. Coughlin J.M. Ju J. D. X. Wang B. biosynthesis in an type I polyketide synthase that two Biol. Chem. PubMed Scopus Google Scholar). OzmQ-KS1 showed a dimeric state in aqueous TAS1 KS (Fig. that the monomeric state is characteristic of TAS1 KS. To the unique TAS1 KS domain that has cyclization in the course of TeA we its crystal structure and to that of previously reported KS TAS1 KS domain was and its structure at resolution (Fig. The TAS1 KS domain shows a typical of KS domain structures Ames B.D. enzymology of polyketide Enzymol. 2009; PubMed Scopus Google Scholar). the overall structure of the TAS1 KS domain was to that of the type I PKS KS domain Y. Cane D.E. Khosla C. The crystal structure of a homodimeric of the 2006; PubMed Scopus Google Scholar), with only ketide extension of to that of the type III PKS domain T. D. T. H. for the formation of from two by the fungal type III polyketide Biol. Chem. 2015; PubMed Scopus Google Scholar), with ketide extension and cyclization of (Fig. that the TAS1 KS domain is to type I PKS KS domain as a independent in analysis (1Yun C.-S. Motoyama T. Osada H. Biosynthesis of the mycotoxin tenuazonic acid by a fungal NRPS-PKS hybrid enzyme.Nat. Commun. 2015; 6 (26503170): 875810.1038/ncomms9758Crossref PubMed Scopus (79) Google Scholar), and the crystal structure this structures of TAS1 KS and type I PKS KS from synthase Y. Cane D.E. Khosla C. The crystal structure of a homodimeric of the 2006; PubMed Scopus Google Scholar) considerable for the structure the substrate-binding which is only present in the type I PKS KS structure (Fig. and Type I PKS KS crystal structures without this have been and KS domains are all the NRPS that KS domains amino acid–containing substrates J.L. G.R. The polyketide synthase into a biosynthetic Biol. PubMed Scopus Google Scholar, J.R. J. B. Y. C. J. H. G. A. B. and of type I polyketide synthase 2015; PubMed Scopus Google Scholar) (Fig. of this structure be a of KS domains that substrates which amino acid. pocket structure of the TAS1 KS is to that of type I PKS KS. The catalytic triad of type I PKS KS domain is The structure of type I polyketide Prod. Rep. 2012; PubMed Scopus Google Scholar), and TAS1 KS has a (Fig. To the catalytic role of the catalytic triad in cyclization, we the of KS The not show (Fig. This is not from the acyl carrier to the catalytic residue of KS is the of KS in PKS T. J. Cane D.E. Khosla C. of active in the ketosynthase domain of an polyketide synthase.Biochemistry. PubMed Scopus (33) Google Scholar). the that in the is to and that the not the for and to 6 and of KS, we of is in the to the catalytic triad and not TAS1 KS and type I PKS KS, is a (Fig. the which also to of KS (Fig. These that the are for the of TAS1 KS. TAS1 KS shows two of with type I PKS KS, His to and to To determine whether cyclization, we and single and The and and the showed and with (Fig. The that and are not essential for the cyclization of TAS1 KS. The secondary structure of all the in this was confirmed using analysis (Fig. To a insight into the substrate and the key amino we of TAS1 KS and an (1Yun C.-S. Motoyama T. Osada H. Biosynthesis of the mycotoxin tenuazonic acid by a fungal NRPS-PKS hybrid enzyme.Nat. Commun. 2015; 6 (26503170): 875810.1038/ncomms9758Crossref PubMed Scopus (79) Google we not obtain a we in using the crystallographic structure of TAS1 KS as a and to as substrate the we the of the with the this the substrate was the two catalytically and and a bond the of the moiety of the and the of residue (Fig. and Because the of His-322 is to the methylene moiety of the His-322 the methylene proton to a that is by two (Fig. and on the crystal structure and of TAS1 KS, we that is by to and the methylene proton abstraction by His-322 a on the to the tetramic acid ring and TeA (Fig. To from the we the not have proton or and is not The show that was to TeA (Fig. However, we the that the chain of substrate TeA To we a analysis to The substrate was for analysis of TAS1 KS domain in report (1Yun C.-S. Motoyama T. Osada H. Biosynthesis of the mycotoxin tenuazonic acid by a fungal NRPS-PKS hybrid enzyme.Nat. Commun. 2015; 6 (26503170): 875810.1038/ncomms9758Crossref PubMed Scopus (79) Google Scholar). However, we and using this substrate the substrate KS domain as a we synthesized an of the substrate moiety of active methylene moiety that abstraction for cyclization and product and complexes in and whereas no was observed in These show the that the can but not the substrate (Fig. and that proton abstraction from the active methylene moiety is a for cyclization. We also which is the of the catalytic pocket in TAS1 KS (Fig. the showed TeA We that the in be of a resolution of an a of the substrate and a residue or of from the residue. To we with and amino acid at and These showed TeA to that resolution of the is not the for increased of (Fig. We also with and amino acid and was as active as but produced TeA (Fig. that of the from increased KS To substrate on the analysis was The to the of was that of (Fig. is that the increased of is related to the was and for and and the for was an with which was (Fig. However, the mechanism the increased of remains using crystal structure analysis of of TAS1 with the substrate be to the mechanism from to the KS domain. The amino ketosynthase domain of shows substrate specificity at the of the amino acid–containing substrate C. A. J. ketosynthase domain from a polyketide synthase for predicted 2013; Scopus Google Scholar). we predicted that of the in the in the of substrates with amino To the and synthesized and The show that substrates by (Fig. 6 and and KS also the (Fig. TeA and of reported in the of TeA S. J. of tenuazonic acid PubMed Scopus Google Scholar). We also amino as substrates with the full-length TAS1 enzyme (1Yun C.-S. Motoyama T. Osada H. Biosynthesis of the mycotoxin tenuazonic acid by a fungal NRPS-PKS hybrid enzyme.Nat. Commun. 2015; 6 (26503170): 875810.1038/ncomms9758Crossref PubMed Scopus (79) Google Scholar) and the product was only in amino (Fig. This suggests that TAS1 KS domain can at the amino as as the and that TAS1 KS has for a broad of A of tetramic acid natural products have been to in and and have a of such as and X. Ju J. tetramic acid structure and biological 2014; Google Scholar). To enzymes or domains for tetramic acid ring formation have been in to TAS1 KS. tetramic acid–containing natural products are biosynthesized by a PKS–NRPS hybrid and tetramic acid ring structure formation is related to the terminal cyclization domain that has a to X. Walsh C.T. acid biosynthesis in of a reductase-like domain in synthetase that and 2009; 48 PubMed Scopus Google Scholar, J.W. role for fungal PKS-NRPS hybrid Am. Chem. Soc. 2008; PubMed Scopus (88) Google Scholar). tetramic acid secondary metabolites are biosynthesized by PKS–NRPS hybrid enzymes or of type I PKS and NRPS modular and tetramic acid ring formation the domain at the of the PKS–NRPS Dieckmann cyclization in and biosynthesis G. Y. J. H. K. Y. Y. F. Biosynthesis of a tetramic from Am. Chem. Soc. PubMed Scopus Google Scholar, S. R. J. biosynthetic for from PubMed Scopus Google Scholar, G. Zhang Zhang T. Y. S. Zhang H. R. Zhang C. into formation by cyclization in Chem. Int. Ed. 2014; PubMed Scopus Google Scholar). tetramic acid ring formation the of Dieckmann was reported in biosynthesis C. X. X. J. Ju J. of a of Dieckmann essential to tetramic acid and natural products 2015; PubMed Scopus Google Scholar). in the course of two that to of complexes and to the are to be in tetramic acid ring formation X. into biosynthesis a for Am. Chem. Soc. 2012; PubMed Scopus Google Scholar). However, the molecular mechanism of all the reported enzymes and TAS1 KS, that catalyze tetramic acid ring formation is this we one of the mechanism of tetramic acid ring formation in natural product The TAS1 KS domain showed a monomeric state in to type which are present in a dimeric KS domain from the multidomain PKS show that the dimeric feature of the KS domain is for dimerization of the full-length PKS A. A. Smith of the of the acid of the 2004; PubMed Scopus Google Scholar). we not the association state of the of the KS domain in this enzyme is from KSs. the role of His-322 and in the cyclization was However, the role of in the course of substrate cyclization remains in this TeA has peptide and we that by His-322 to and a of the on the to TeA (Fig. using the as a show the of the substrate the to the and and is for (Fig. The residue to the of the for the to amino not the or To the role of we two His-322 and We that be of amino His and that be of amino we predicted that not substrate amino not we also to the but we not to the The the of protein, but at this we not the was The not TeA its to the However, a TeA not proton abstraction These that proton abstraction from the substrate by His-322 is not the mechanism for substrate cyclization. be out that is and to has a substrate-binding pocket by substitution of amino and we observed that the substrate is in aqueous solution. cyclization in the of by amino acid in the substrate-binding Because of the structural diversity of natural products using domain in PKS and NRPS has been for polyketide synthase Prod. Rep. 2018; 35 PubMed Google Scholar, F. A. F. A. and of peptide Chem. 2018; PubMed Google Scholar, T. T. Zhang S. J. H. K. A. of the NRPS-PKS by gene Commun. 2018; PubMed Scopus Google Scholar). acid–containing substrate-accepting have substrate C. A. J. ketosynthase domain from a polyketide synthase for predicted 2013; Scopus Google Scholar), which domain polyketide synthase and nonribosomal peptide Opin. Biol. 2013; 23 PubMed Scopus Google Scholar). that PKS systems that amino into products be to using TAS1 KS a domain with broad substrate this we a mechanism of substrate cyclization by the KS domain in TAS1 with its unique features and an to the structural diversity of natural products of polyketide and peptide using TAS1 KS. and from and and from of and in this are in was in at and was performed to J. T. Molecular A Scholar). was out using or to and for and performed to J. T. Molecular A Scholar). was performed with a was for via from using a for was out using an from and with a using an with a The KS domain of TAS1 was from (1Yun C.-S. Motoyama T. Osada H. Biosynthesis of the mycotoxin tenuazonic acid by a fungal NRPS-PKS hybrid enzyme.Nat. Commun. 2015; 6 (26503170): 875810.1038/ncomms9758Crossref PubMed Scopus (79) Google Scholar) via using TAS1 and TAS1 and the was and into the that using and the and the termination via to construct the KS KS using the the which a to at the and TAS1 to the 1 KS from the to the residue from The and to the and the KS from to the termination from 1 and using the to yield and into The of all was confirmed by and to the gene from by J.R. J. B. Y. C. J. H. G. A. B. and of type I polyketide synthase 2015; PubMed Scopus Google Scholar). The was and into the for TAS1 KS The in this are in To KS protein, the was into at in with 1 was into with and at for the via for The in 1 and and on for with was via for at The was on a acid as previously (1Yun C.-S. Motoyama T. Osada H. Biosynthesis of the mycotoxin tenuazonic acid by a fungal NRPS-PKS hybrid enzyme.Nat. Commun. 2015; 6 (26503170): 875810.1038/ncomms9758Crossref PubMed Scopus (79) Google Scholar). The was with protein) at for to was with to and was with acid from the KS KS expressed and using the at The C-A-PCP domain was expressed using a as previously (1Yun C.-S. Motoyama T. Osada H. Biosynthesis of the mycotoxin tenuazonic acid by a fungal NRPS-PKS hybrid enzyme.Nat. Commun. 2015; 6 (26503170): 875810.1038/ncomms9758Crossref PubMed Scopus (79) Google Scholar). TAS1 KS domain was to in 1 using a was performed using the was by 1 of TAS1 KS domain solution and solution acid at within in and using a at K. at at to for and and using and from the Crystal structure was with molecular using Molecular solution was from KS domain that is of A polyketide synthase J.R. S. Hansen D.A. Sherman D.H. Smith polyketide synthase A for natural product Biol. 2013; PubMed Scopus Google Scholar) as a and performed using and The overall in the structure was with of in of the and only one The and are in Molecular in this using TeA with TAS1 KS and C-A-PCP domain was at for in 1 KS protein, C-A-PCP domain synthesized in a 1 from P. 1 1 and 1 analysis of substrate cyclization and or at for To determine the the of from to The and using to the to the using the enzyme performed in the enzyme the with of with an and in for analysis of TeA analysis was out with an with a The as A, the into a with with a linear from to the course of and at for in the and of by with was for was for on a in with as and in on a in as and and in The to the resolution on a of to a was from using a D.E. J.W. T. Walsh C.T. as substrates for the condensation domains of nonribosomal peptide Biol. 2000; 7 PubMed Scopus Google Scholar). A solution of in was at a A solution of and in and was the was for 1 was and the was for The was and the residue was in The was with aqueous solution and and The product was using chromatography to as a The derivative was in and The was for 1 at and The residue was in and to to obtain A solution of in was at a and The was for at with aqueous and The residue was in The was with and The product was with to provide as a for was from via using the for a a was from using the for a for A solution of (1Yun C.-S. Motoyama T. Osada H. Biosynthesis of the mycotoxin tenuazonic acid by a fungal NRPS-PKS hybrid enzyme.Nat. Commun. 2015; 6 (26503170): 875810.1038/ncomms9758Crossref PubMed Scopus (79) Google Scholar) and acid of Chem. Scopus Google Scholar, S. of via to dimerization of 2013; Scopus Google Scholar) in was at a and and the was for 1 the was The residue was by chromatography to 6 as a for The of the and from the and The secondary structure of all the and in this was at from to as the of using in a at a of and a of 1 all was The TAS1 KS was with and and to in an with a of The for was with the and to These using chromatography at with a and at molecular and in and also in with and KS at for the using with a A acid solution in was A acid solution was also as A solution was with of solution. A of was with of the solution and at on the for PKS KS the substrate of the TAS1 KS domain be and Dieckmann cyclization to tenuazonic acid. To for an of the substrate to was to to a bond in and was using as The structures of and and with The of was with and the moiety of the substrate was with the chain of to a bond using to the and and using The was to analysis was performed at the of the that within a of in the and clusters to The and structure of the reported crystal structures have been in the The that all the findings of this are within the and its The was by B. We T. for We and for with tenuazonic acid nonribosomal peptide synthetase polyketide synthase ketosynthase thioesterase peptidyl carrier chromatography

Topics & Concepts