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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Abstract
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 |
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| 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 |
| Refereed: | Yes |
| 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: | 1433-7851 |
| 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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