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

Abstract

S-Adenosyl methionine (SAM)-dependent C-methyltransferases are responsible for the C2-methylation of 3-ketoacyl-acyl carrier protein (ACP) intermediates to give the corresponding 2-methy-3-ketoacyl-ACP products during bacterial polyketide biosynthesis mediated by trans-AT polyketide synthases that lack integrated acyl transferase (AT) domains. A coupled ketoreductase (KR) assay was used to assign the stereochemistry of the C-methyltransferase-catalyzed reaction. Samples of chemoenzymatically-generated 3-ketopentanoyl-ACP (9) were incubated with SAM and BonMT2 from module 2 of the bongkrekic acid polyketide synthase. The resulting 2-methyl-3-ketopentanoyl-ACP (10) was incubated separately with five (2R)- or (2S)-methyl specific KR domains. Analysis of the derived 2-methyl-3-hydroxypentanoate methyl esters (8) by chiral GC-MS established that the BonMT2-catalyzed methylation generated exclusively (2R)-2-methyl-3-ketopentanoyl-ACP ((2R)-10). Identical results were also obtained with three additional C-methyltransferases – BaeMT9, DifMT1, and MupMT1 – from the bacillaene, difficidin, and mupirocin trans-AT polyketide synthases

A 3‐hydroxy‐3‐methylglutaryl‐CoA synthase‐based probe for the discovery of the acyltransferase‐less type I polyketide synthases

“…Polyketides produced by AT-less type I PKSs represent a growing family of natural products with complex molecular structures and promising biological activities (Piel, 2010;Helfrich and Piel, 2016). Compared with canonical type I PKSs, AT-less type I PKSs possess not only KS domains specific for various alkyl substrates in individual modules, but also various tailoring enzymes for online modification of the growing polyketide chains, such as the embedded methyltransferase, DUF-SH domains and HCSs (Pan et al, 2017;Xie et al, 2017). HCSs play important roles to diversify polyketide structures for modular PKSs, especially AT-less type I PKSs (Calderone et al, 2006).…”

A 3-hydroxy-3-methylglutaryl-CoA synthase-based probe for the discovery of the acyltransferase-less type I polyketide synthases

“…Polyketides produced by AT-less type I PKSs represent a growing family of natural products with complex molecular structures and promising biological activities (Piel, 2010;. Compared to canonical type I PKSs, AT-less type I PKSs possess not only KS domains specific for various alky substrates in individual modules, but various tailoring enzymes for online modification of the growing polyketide chains, such as the imbedded methyltransferase, DUF-SH domains and HCSs (Pan et al, 2017;Xie et al, 2017). HCSs play important roles to diversify polyketide structures for modular PKSs, especially AT-less type I PKSs (Calderone et al, 2006).…”

Synthetic biology of polyketide synthases

“…On the other hand, the C-MT domain of the PKS module for yersiniabactin biosynthesis specifies dimethylation to produce the corresponding gem-dimethyl group [37]. Recently, C-MTs from trans-AT PKS systems have been shown to be stereospecific [50].…”