Trapping of a Polyketide Synthase Module after C−C Bond Formation Reveals Transient Acyl Carrier Domain Interactions

authored by: Maria Dell, Mai Anh Tran, Michael J. Capper, Srividhya Sundaram , Jonas Fiedler, Jesko Koehnke, Ute A. Hellmich, Christian Hertweck
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.
Organisation(s): Institute of Food Chemistry
External Organisation(s): Leibniz Institute for Natural Product Research and Infection Biology Hans Knöll Institute (HKI)
Friedrich Schiller University Jena
University of Glasgow
Goethe University Frankfurt
Type: Article
Journal: Angewandte Chemie - International Edition
Volume: 63
No. of pages: 8
ISSN: 1433-7851
Publication date: 19.02.2024
Publication status: Published
Peer reviewed: Yes
ASJC Scopus subject areas: Catalysis, General Chemistry
Electronic version(s): https://doi.org/10.1002/anie.202315850 (Access: Open)

BibTeX

@article{934b66019d484c67bb6b627d37f87609,
title = "Trapping of a Polyketide Synthase Module after C−C Bond Formation Reveals Transient Acyl Carrier Domain Interactions",
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.",
keywords = "Acyl Carrier Protein, Biosynthesis, Crosslinking, Electron Microscopy, Modular Polyketide Synthases",
author = "Maria Dell and Tran, {Mai Anh} and Capper, {Michael J.} and Srividhya Sundaram and Jonas Fiedler and Jesko Koehnke and Hellmich, {Ute A.} and Christian Hertweck",
note = "Funding Information: This work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany's Excellence Strategy—EXC 2051—Project-ID 390713860, CRC 1127—Project-ID 239748522 (ChemBioSys) (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 (ERC CoG 101002326) (to J.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-EU EFRE 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. ",
year = "2024",
month = feb,
day = "19",
doi = "10.1002/anie.202315850",
language = "English",
volume = "63",
journal = "Angewandte Chemie - International Edition",
issn = "1433-7851",
publisher = "John Wiley and Sons Ltd",
number = "9",
}