Molecular Basis for Short-Chain Thioester Hydrolysis by Acyl Hydrolases in trans-Acyltransferase Polyketide Synthases

Polyketide synthases (PKSs) are multidomain enzymatic assembly lines that biosynthesize a wide selection of bioactive natural products from simple building blocks. In contrast to their cis-acyltransferase (AT) counterparts, trans-AT PKSs rely on stand-alone ATs to load extender units onto acyl carrier protein (ACP) domains embedded in the core PKS machinery. Trans-AT PKS gene clusters also encode stand-alone acyl hydrolases (AHs), which are predicted to share the overall fold of ATs but function like type II thioesterases (TE_IIs), hydrolyzing aberrant acyl chains from ACP domains to promote biosynthetic efficiency. How AHs specifically target short acyl chains, in particular acetyl groups, tethered as thioesters to the substrate-shuttling ACP domains, with hydrolytic rather than acyl transfer activity, has remained unclear. To answer these questions, we solved the first structure of an AH and performed structure-guided activity assays on active site variants. Our results offer key insights into chain length control and selection against coenzyme A-tethered substrates, and clarify how the interaction interface between AHs and ACP domains contributes to recognition of cognate and noncognate ACP domains. Combining our experimental findings with molecular dynamics simulations allowed for the construction of a data-driven model of an AH:ACP domain complex. Our results advance the currently incomplete understanding of polyketide biosynthesis by trans-AT PKSs, and provide foundations for future bioengineering efforts to offload biosynthetic intermediates or enhance product yields.