Mechanism and Stereochemistry of Polyketide Chain Elongation and Methyl Group Epimerization in Polyether Biosynthesis

Abstract

The polyketide synthases responsible for the biosynthesis of the polyether antibiotics nanchangmycin (1) and salinomycin (4) harbor a number of redox-inactive ketoreductase (KR0) domains that are implicated in the generation of C2-epimerized (2S)-2-methyl-3-ketoacyl-ACP intermediates. Evidence that the natural substrate for the polyether KR0 domains is, as predicted, a (2R)-2-methyl-3-ketoacyl-ACP intermediate, came from a newly developed coupled ketosynthase (KS)-ketoreductase (KR) assay that established that the decarboxylative condensation of methylmalonyl-CoA with S-propionyl-N-acetylcysteamine catalyzed by the Nan[KS1][AT1] didomain from module 1 of the nanchangmycin synthase generates exclusively the corresponding (2R)-2-methyl-3-ketopentanoyl-ACP (7a) product. In tandem equilibrium isotope exchange experiments, incubation of [2-2H]-(2R,3S)-2-methyl-3-hydroxypentanoyl-ACP (6a) with redox-active, epimerase-inactive EryKR6 from module 6 of the 6-deoxyerythronolide B synthase and catalytic quantities of NADP+ in the presence of redox-inactive, recombinant NanKR10 or NanKR50, from modules 1 and 5 of the nanchangmycin synthase, or recombinant SalKR70 from module 7 of the salinomycin synthase, resulted in first-order, time-dependent washout of deuterium from 6a. Control experiments confirmed that this washout was due to KR0-catalyzed isotope exchange of the reversibly-generated, transiently-formed oxidation product [2-2H]-(2R)-2-methyl-3-ketopentanoyl-ACP (7a), consistent with the proposed epimerase activity of each of the KR0 domains. Although they belong to the superfamily of short chain dehydrogenase-reductases, the epimerase-active KR0 domains from polyether synthases lack one or both residues of the conserved Tyr-Ser dyad that has previously been implicated in KR-catalyzed epimerizations.

Substrate-bound structures of a ketoreductase from amphotericin modular polyketide synthase

“…Complex polyketides harbor numerous methyl groups possessing L stereochemistry, whereas the KSs of modular PKSs catalyze the generation of (2D)-2-methyl-3-ketoacyl-ACPs exclusively during the elongation of polyketide intermediates (Castonguay et al, 2007; Weissman et al, 1997; Xie et al, 2017). In addition, the binding of the acyl group to a patch on the surface of the ACP prevents the water-catalyzed epimerization observed for the C2 methyl group of the NAC- or pantetheine-bound thioesters (Castonguay et al, 2007; Vance et al, 2016).…”

“…The EIX assays provide direct evidence that A2- and B2-type KRs have intrinsic epimerase activities. Disappointingly, the AmpKR2 G355T/Q364H mutant failed to exhibit any detectable epimerase activity in the EIX assay, suggesting that the introduced double mutations only alter the diastereoselectivity of AmpKR2 (Xie et al, 2017). Site-directed mutagenesis of epimerase-active KRs suggests the epimerization reaction utilizes the same catalytic residues as the ketoreduction reaction.…”

Stereospecific Formation of E- and Z-Disubstituted Double Bonds by Dehydratase Domains from Modules 1 and 2 of the Fostriecin Polyketide Synthase

“…30 Growth media and conditions used for E. coli strains and standard methods for handling E. coli in viv o and in vitro were those described previously, unless otherwise noted. 31 All DNA manipulations were performed following standard procedures.…”

Elucidation of the Stereospecificity of C-Methyltransferases from trans-AT Polyketide Synthases

“…6b Using this assay, we established unambiguously that the decarboxylative condensation catalyzed by the NanKS1 domain of nanchangmycin synthase generates exclusively a (2 R )-2-methyl-3-ketoacyl-ACP product. In fact, the coupled KR-assay is entirely general and can be used to assign the stereochemistry of any chemoenzymatically generated 2-methyl-3-ketoacyl-ACP.…”

“…Incubation of BonMT2 with the N -acetylcysteamine thioester 3-ketopentanoyl-SNAC ( 5 ) and SAM, in the presence of S -adenosyl homocysteine (SAH) nucleosidase to relieve potent product inhibition by SAH, 8,10 gave 2-methyl-3-ketopentanoyl-SNAC ( 6 ), as established by HPLC-MS (Scheme 1, Figure S3). The product was further characterized by reduction with TylKR1 from module 1 of the tylactone synthase 6,7b,7e in the presence of NADPH to give (2 R ,3 R )-2-methyl-3-hydroxypentanoyl-SNAC ( 7a ), whose structure and stereochemistry were established by base-catalyzed thioester hydrolysis and treatment with trimethylsilyldiazomethane (TMS-CHN 2 ). Chiral GC-MS analysis of the derived methyl esters confirmed predominant formation of the characteristic reduction product, methyl (2 R ,3 R )-2-methyl-3-hydroxypentanoate ( 8a ) (Figure S4).…”