Trapping of a polyketide synthase module after C−C bond formation reveals transient acyl carrier domain interactions

Dell, M., Tran, M. A., Capper, M. J., Sundaram, S., Fiedler, J., Koehnke, J. , Hellmich, U. A. and Hertweck, C. (2023) Trapping of a polyketide synthase module after C−C bond formation reveals transient acyl carrier domain interactions. Angewandte Chemie (International Edition), (doi: 10.1002/anie.202315850) (PMID:38134222) (Early Online Publication)

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Modular polyketide synthases (PKSs) are giant assembly lines that produce an impressive range of biologically active compounds. However, our understanding of the structural dynamics of these megasynthases, specifically the delivery of acyl carrier protein (ACP)‐bound building blocks to the catalytic site of the ketosynthase (KS) domain, remains severely limited. Using a multipronged structural approach, we report details of the inter‐domain interactions after C−C bond formation in a chain‐branching module of the rhizoxin PKS. Mechanism‐based crosslinking of an engineered module was achieved using a synthetic substrate surrogate that serves as a Michael acceptor. The crosslinked protein allowed us to identify an asymmetric state of the dimeric protein complex upon C−C bond formation by cryo‐electron microscopy (cryo‐EM). The possible existence of two ACP binding sites, one of them a potential “parking position” for substrate loading, was also indicated by AlphaFold2 predictions. NMR spectroscopy showed that a transient complex is formed in solution, independent of the linker domains, and photochemical crosslinking/mass spectrometry of the standalone domains allowed us to pinpoint the interdomain interaction sites. The structural insights into a branching PKS module arrested after C−C bond formation allows a better understanding of domain dynamics and provides valuable information for the rational design of modular assembly lines.

Item Type:Articles
Additional Information:This work was funded by the DeutscheFor-schungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy—EXC2051—Project-ID390713860, CRC1127—Project-ID239748522 (Chem-BioSys)(to U.A.H .and C.H.), and Leibniz Award (to C.H.), by the European Regional Development Fund (ERDF) (MassNat) (to C.H.), and the European Research Council (ERCCoG101002326) (toJ.K.). We acknowledge the Scottish Centre for Macromolecular Imaging (SCMI) and James Streetley for assistance with cryo-EM experiments and access to instrumentation, funded by the MRC (MC_PC_17135) and SFC (H17007). U.A.H. acknowledges an instrumentation grant for a high-field NMR spectrometer by the REACT-EUEFRE Thuringia (Recovery assistance for cohesion and the territories of Europe, ERDF, Thuringia) initiative of the European Union. Open Access funding enabled and organized by Projekt DEAL.
Status:Early Online Publication
Glasgow Author(s) Enlighten ID:Capper, Dr Michael and Koehnke, Professor Jesko
Authors: Dell, M., Tran, M. A., Capper, M. J., Sundaram, S., Fiedler, J., Koehnke, J., Hellmich, U. A., and Hertweck, C.
College/School:College of Science and Engineering > School of Chemistry
Journal Name:Angewandte Chemie (International Edition)
Publisher:Wiley - V C H Verlag GmbH & Co. KGaA
ISSN (Online):1521-3773
Published Online:22 December 2023
Copyright Holders:Copyright © 2023 The Authors
First Published:First published in Angewandte Chemie (International Edition) 2023
Publisher Policy:Reproduced under a Creative Commons licence

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
310456Exploitation of enzyme promiscuity to generate ribosomal natural product diversityJesko KoehnkeEuropean Commission (EC)101002326Chemistry
301445The Scottish Macromolecular Imaging Centre (SMIC)David BhellaMedical Research Council (MRC)MC_PC_17135SII - Virology
301474The purchase and maintenance of a cryo-electron microscopeDavid BhellaScottish Funding Council (SFC)H17007SII - Virology