Details
| Original language | English |
|---|---|
| Article number | e202315850 |
| Number of pages | 8 |
| Journal | Angewandte Chemie - International Edition |
| Volume | 63 |
| Issue number | 9 |
| Early online date | 22 Dec 2023 |
| Publication status | Published - 19 Feb 2024 |
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
ASJC Scopus subject areas
- Chemical Engineering(all)
- Catalysis
- Chemistry(all)
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In: Angewandte Chemie - International Edition, Vol. 63, No. 9, e202315850, 19.02.2024.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Trapping of a Polyketide Synthase Module after C−C Bond Formation Reveals Transient Acyl Carrier Domain Interactions
AU - Dell, Maria
AU - Tran, Mai Anh
AU - Capper, Michael J.
AU - Sundaram , Srividhya
AU - Fiedler, Jonas
AU - Koehnke, Jesko
AU - Hellmich, Ute A.
AU - Hertweck, Christian
N1 - 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.
PY - 2024/2/19
Y1 - 2024/2/19
N2 - 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.
AB - 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.
KW - Acyl Carrier Protein
KW - Biosynthesis
KW - Crosslinking
KW - Electron Microscopy
KW - Modular Polyketide Synthases
UR - http://www.scopus.com/inward/record.url?scp=85182415175&partnerID=8YFLogxK
U2 - 10.1002/anie.202315850
DO - 10.1002/anie.202315850
M3 - Article
C2 - 38134222
AN - SCOPUS:85182415175
VL - 63
JO - Angewandte Chemie - International Edition
JF - Angewandte Chemie - International Edition
SN - 1433-7851
IS - 9
M1 - e202315850
ER -