α-Methylation follows condensation in the gephyronic acid modular polyketide synthase

Abstract

C-methyltransferases (MTs) from modular polyketide synthase assembly lines are relatively rare and unexplored domains that are responsible for installing α-methyl groups into nascent polyketide backbones. The stage at which these synthase-embedded enzymes operate during polyketide biosynthesis has yet to be conclusively demonstrated. In this work we establish the activity and substrate preference for six MTs from the gephyronic acid polyketide synthase and demonstrate their ability to methylate both N-acetylcysteamine- and acyl carrier protein-linked β-ketoacylthioester substrates but not malonyl thioester equivalents. These data strongly indicate that MT-catalyzed methylation occurs immediately downstream of ketosynthase-mediated condensation during polyketide assembly. This work represents the first successful report of MT-catalyzed mono- and dimethylation of simple thioester substrates and provides the groundwork for future mechanistic and engineering studies on this important but poorly understood enzymatic domain.

Domain Organization and Active Site Architecture of a Polyketide Synthase C-methyltransferase

“…Thus it was unclear when monomethylation occurs in modular C -MTs. Recent work demonstrated C -MT activity via Route 1 in bacterial trans -AT and cis -AT pathways 24, 25 .…”

Leveraging microbial biosynthetic pathways for the generation of ‘drop-in’ biofuels

“…Methylation through SAM-dependent C -methyltransferases is an alternative approach that has not been extensively studied. Recent studies have helped to elucidate the reaction mechanism of methyltransferases, indicating that methylation can occur before or after Claisen condensation [56,57], and also that excised mono- and di-methyltransferases can function in vitro [57,58]. This exquisite control over product formation could generate customized products with desired combustion properties as biofuel substitutes.…”

Reversible Product Release and Recapture by a Fungal Polyketide Synthase Using a Carnitine Acyltransferase Domain

“…This is consistent with the suggestion of four SAM-derived methyl group from 13 C labeling studies described above. The gem -dimethyl group in polyketides are relatively rare, most examples are from bacterial polyketides, [13] including the anticancer drugs epothilone and precursor of epoxyketone. [14] Keasling and coworkers showed that MT domains in these PKSs can gem- dimethylate either the β-keto polyketide intermediate, or malonyl-ACP prior to KS-catalyzed condensation.…”

“…[14] Keasling and coworkers showed that MT domains in these PKSs can gem- dimethylate either the β-keto polyketide intermediate, or malonyl-ACP prior to KS-catalyzed condensation. [13h] Keatinge-Clay and coworkers showed the GphH MT domain from the gephyronic acid biosynthetic pathway can gem- dimethylate acetoacetyl- S-N- acetylcysteamine (∼1 turnover in 72 hours). [13i] Fungal polyketides containing gem -dimethyl groups are rarer, [15] with no responsible MT identified so far.…”

“…[13h] Keatinge-Clay and coworkers showed the GphH MT domain from the gephyronic acid biosynthetic pathway can gem- dimethylate acetoacetyl- S-N- acetylcysteamine (∼1 turnover in 72 hours). [13i] Fungal polyketides containing gem -dimethyl groups are rarer, [15] with no responsible MT identified so far.…”