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The structural organization of substrate loading in iterative polyketide synthases

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Abstract

Polyketide synthases (PKSs) are microbial multienzymes for the biosynthesis of biologically potent secondary metabolites. Polyketide production is initiated by the loading of a starter unit onto an integral acyl carrier protein (ACP) and its subsequent transfer to the ketosynthase (KS). Initial substrate loading is achieved either by multidomain loading modules or by the integration of designated loading domains, such as starter unit acyltransferases (SAT), whose structural integration into PKS remains unresolved. A crystal structure of the loading/condensing region of the nonreducing PKS CTB1 demonstrates the ordered insertion of a pseudodimeric SAT into the condensing region, which is aided by the SAT-KS linker. Cryo-electron microscopy of the post-loading state trapped by mechanism-based crosslinking of ACP to KS reveals asymmetry across the CTB1 loading/–condensing region, in accord with preferential 1:2 binding stoichiometry. These results are critical for re-engineering the loading step in polyketide biosynthesis and support functional relevance of asymmetric conformations of PKSs.

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Fig. 1: Domain organization and catalytic scheme of the cercosporin PKS CTB1.
Fig. 2: Crystal structure and interdomain interactions in CTB1 SAT-KS-MAT.
Fig. 3: Asymmetric cryo-EM structure of CTB1 SAT°-KS-MAT°=ACP2.
Fig. 4: Schematic illustration of suggested modes of conformational coupling in CTB1.

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  • 10 April 2018

    In the version of this article originally published, “Supplementary Text and Figures” in the Supplementary Information section incorrectly linked to the Life Sciences Reporting Summary instead of the file containing Supplementary Tables 1–4 and Supplementary Figures 1–12. The error has been corrected in the HTML version of this article.

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Acknowledgements

We thank P. Leadlay and L. Betancor for providing plasmid pETcoco-2A-L1SL2. X-ray diffraction data were collected at beamline PXI of the Paul Scherrer Institute, Villigen, Switzerland, and cryo-EM data were collected at the BioEM facility of the University of Basel; we acknowledge excellent support by teams of both facilities. This work was supported by the Swiss National Science Foundation (SNF) project grants 138262, 159696, SNF R’equip grants 145023 and 164074, and the National Institutes of Health (ES001670). D.A.H. acknowledges a fellowship from the Werner-Siemens Foundation.

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Authors and Affiliations

  1. Department of Biozentrum, University of Basel, Basel, Switzerland

    Dominik A. Herbst, Roman P. Jakob & Timm Maier

  2. Department of Chemistry, Johns Hopkins University, Baltimore, MD, USA

    Callie R. Huitt-Roehl, Jacob M. Kravetz, Philip A. Storm, Jamie R. Alley & Craig A. Townsend

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  1. Dominik A. Herbst
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Contributions

R.P.J. expressed, purified and crystallized CTB1 SAT-KS-MAT. D.A.H., R.P.J., and T.M. solved the crystal structure. D.A.H. performed cryo-EM, data processing, modeling, refinement and analysis of all structural data. C.R.H.-R. optimized and prepared crosslinked CTB1 SAT°-KS-MAT°=ACP2 for structural analysis and performed mutational experiments for structural validation. J.M.K. synthesized the α-bromopropionyl crosslinker. P.A.S. and J.R.A. performed initial exploratory experiments, and J.R.A. prepared the CTB1 SAT°-KS-MAT° construct for crosslinking. C.A.T. and T.M. designed research. The manuscript was written by D.A.H., T.M., C.R.H.-R., and C.A.T.

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Correspondence to Timm Maier.

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Supplementary Tables 1–4 and Supplementary Figures 1–12

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Supplementary Note 1:

Synthetic Procedures

Supplementary Video 1

ACP2 binding to the CTB1 loading/condensing region

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Herbst, D.A., Huitt-Roehl, C.R., Jakob, R.P. et al. The structural organization of substrate loading in iterative polyketide synthases. Nat Chem Biol 14, 474–479 (2018). https://doi.org/10.1038/s41589-018-0026-3

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