Polyketide Proofreading by an Acyltransferase-like Enzyme

Abstract

SUMMARY Trans-acyltransferase polyketide synthases (trans-AT PKSs) are an important group of bacterial enzymes producing bioactive polyketides. One difference from textbook PKSs is the presence of one or more free-standing AT-like enzymes. While one homolog loads the PKS with malonyl units, the function of the second copy (AT2) was unknown. We studied the two ATs PedC and PedD involved in pederin biosynthesis in an uncultivated symbiont. PedD displayed malonyl- but not acetyltransferase activity toward various acyl carrier proteins (ACPs). In contrast, the AT2 PedC efficiently hydrolyzed acyl units bound to N-acetylcysteamine or ACP. It accepted substrates with various chain lengths and functionalizations but did not cleave malonyl-ACP. These data are consistent with the role of PedC in PKS proofreading, suggesting a similar function for other AT2 homologs and providing strategies for polyketide titer improvement and biosynthetic investigations.

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“…Other biosynthetic genes are also present, including two hypothetical acyltransferases, trtAB, one of which could act as a proofreader (14); two putative oxidoreductases, trtGI, which appear to encode proteins for oxygenases, and a putative polyketide cyclase, trtJ (15), which may be involved in the cyclization of compound 1. In addition to the integrated thioesterase (TE) in trtF, which is expected to cause release of the elongated chain, a standalone type II TE, trtH, also is present and is proposed to cause regeneration of misprimed thiolation domains (16).…”

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“…S4) also grouped OocV-AT2 with the stand-alone acyltransferase PedC. A recent study showed that PedC does not exhibit AT activity but possesses acyl hydrolase activity and cleaves acyl groups bound to ACP, suggesting a role in PKS biosynthetic proofreading (65).…”

Acyl hydrolases from trans-AT polyketide synthases target acetyl units on acyl carrier proteins

“…However, a recently identified class of trans-acting AT-like domains have been shown to harbour hydrolytic activity towards acylthioesters 8 . These enzymes, subsequently classed as acyl hydrolases (AH), are distinctly different from AT domains at the sequence level and form a separate clade in a phylogenetic analysis of AT and AH domains from trans-AT PKSs (Fig 1 and S8).…”

“…These enzymes, subsequently classed as acyl hydrolases (AH), are distinctly different from AT domains at the sequence level and form a separate clade in a phylogenetic analysis of AT and AH domains from trans-AT PKSs (Fig 1 and S8). AH domains have been proposed to act as proofreading enzymes for the PKS by hydrolysing stalled intermediates from the ACP domains of the biosynthetic assembly line 8,9 . In order to fulfil such a role, the AH domains would require a broad substrate specificity for a range of acyl chains.…”

“…Whilst the latter falls into the AT1 clade with other known malonyl transferases, PedC is located in the largely unstudied AT2 clade (Fig 1 and S8) 8 . An in vitro functional analysis has shown that PedC catalyses the hydrolysis of acyl thioesters derived from both N-acetylcysteamine thioesters (SNACs) and acyl-ACPs, leading to its designation as a proof-reading enzyme that removes stalled polyketide intermediates 8 . A selection of short chain SNACs were previously employed to examine the specificity of PedC, with the 3-hydroxybutyryl, α,β-unsaturated and 4-methylpentanoic species among the better substrates.…”