Thioesterase-mediated O-acetylation and double bond formation in peloruside biosynthesis Amy Fraley , M. Rust, C. L. Dieterich, R. A. Meoded, F. Hemmerling, J. Piel Eidgenössische Technische Hochschule Zürich, Switzerland The sponge Mycale hentscheli is famous for its three distinct anticancer polyketides, among which the tubulin- inhibiting pelorusides exhibit particularly high promise for anticancer drug development, provided that a means of production can be identified. Microbiome sequencing has revealed that these metabolites are biosynthesized by trans -acyltransferase polyketide synthases ( trans -AT PKSs), multimodular megaenzymes responsible for the majority of biosynthetically assigned polyketides from sponge symbionts. While a number have been identified, trans -AT PKSs remain poorly understood from a mechanistic standpoint as they contain a staggering array of novel biosynthetic components. This work started with the assignment of the pel locus to peloruside production based on the knowledge that ketosynthase (KS) sequences can be used to predict partial intermediate structures. However, due to the highly aberrant architecture of modules 2 and 3, the original prediction substantially differed for the exocyclic moiety of the polyketide. These unusual modules feature a series of non-elongating KSs (KS 0 ), a condensation (C) domain that normally occurs in nonribosomal peptide synthetases (NRPSs) to generate amide bonds, and two internal thioesterase (TE) domains, which are usually positioned at the termini of PKSs and NRPSs to catalyze thioester hydrolysis or macrocyclization. We have biochemically characterized this portion of the peloruside biosynthetic pathway and uncovered a novel domain architecture responsible for O -acetylation and double bond formation. Subsequent mining based on KS phylogeny identified homologous systems with similar, yet uncharacterized, enzymology. Through biochemical and structural characterization of a homologous internal TE involved in oocydin biosynthesis, we have provided unique insight into the mechanism of O -acetylation at an early- or late-stage in polyketide biosynthesis.
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