Mechanism of intersubunit ketosynthase–dehydratase interaction in polyketide synthases

Abstract

Modular polyketide synthases (PKSs) produce numerous structurally complex natural products that have diverse applications in medicine and agriculture. PKSs typically consist of several multienzyme subunits that utilize structurally defined docking domains (DDs) at their N and C termini to ensure correct assembly into functional multiprotein complexes. Here we report a fundamentally different mechanism for subunit assembly in trans-acyltransferase (trans-AT) modular PKSs at the junction between ketosynthase (KS) and dehydratase (DH) domains. This mechanism involves direct interaction of a largely unstructured docking domain (DD) at the C terminus of the KS with the surface of the downstream DH. Acyl transfer assays and mechanism-based crosslinking established that the DD is required for the KS to communicate with the acyl carrier protein appended to the DH. Two distinct regions for binding of the DD to the DH were identified using NMR spectroscopy, carbene footprinting, and mutagenesis, providing a foundation for future elucidation of the molecular basis for interaction specificity.

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Figure 1: Bioinformatics analysis of DHD domains and examples from the gladiolin and bacillaene PKSs.
Figure 2: Acyl transfer and protein crosslinking assays demonstrate that DHD domains play a key role in communication across KS/DH interfaces.
Figure 3: NMR titrations reveal two regions of DHD domains involved in the interaction with DH-ACP di-domains.
Figure 4: Mapping DHD and ACP domain interaction sites on the DH domain.
Figure 5: DHD domains interact selectively with their cognate DH domain partners.

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Acknowledgements

This research was supported by grants from the BBSRC (BB/L021692/1 to G.L.C. and BB/L022761/1 to J.R.L.). The Bruker MaXis II instrument used in this study was funded by the BBSRC (BB/M017982/1). N.J.O., J.E.M., L.M. and A.S.B. thank the University of Nottingham for funding. The research leading to these results has received funding from the European Research Council under the European Union's Seventh Framework Programme (FP/2007-2013)/ERC Grant Agreement 639907 (to J.R.L.). G.L.C. is the recipient of a Wolfson Research Merit Award from the Royal Society (WM130033). We thank L. Song for assistance with LC-MS analyses.

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M.J. and G.L.C. designed the study, and all authors contributed to performing the research as follows: M.J. and D.G. carried out bioinformatics analyses; M.J. overproduced and purified all recombinant proteins, and conducted acyl transfer assays and LC–MS analyses of intact proteins; P.P. synthesized the trans-β-chloroacrylamide-containing pantetheine analog, and P.P. and M.J. carried out the crosslinking experiments; S.K. and J.R.L. conducted the NMR and CD experiments, and analyzed the data; A.S.B. and J.E.M. synthesized the diazirine reagent used in the footprinting experiments; L.M. and N.J.O. carried out the footprinting experiments and analyzed the data; M.J., S.K., J.R.L. and G.L.C. wrote the paper, and all authors contributed to revision of the manuscript.

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Correspondence to Matthew Jenner or Gregory L Challis.

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Jenner, M., Kosol, S., Griffiths, D. et al. Mechanism of intersubunit ketosynthase–dehydratase interaction in polyketide synthases. Nat Chem Biol 14, 270–275 (2018). https://doi.org/10.1038/nchembio.2549

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