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Vinylogous chain branching catalysed by a dedicated polyketide synthase module

Nature volume 502, pages 124–128 (2013)Cite this article

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Abstract

Bacteria use modular polyketide synthases (PKSs) to assemble complex polyketides, many of which are leads for the development of clinical drugs, in particular anti-infectives and anti-tumoral agents1. Because these multifarious compounds are notoriously difficult to synthesize, they are usually produced by microbial fermentation. During the past two decades, an impressive body of knowledge on modular PKSs2,3 has been gathered that not only provides detailed insight into the biosynthetic pathways but also allows the rational engineering of enzymatic processing lines to yield structural analogues4,5. Notably, a hallmark of all PKS modules studied so far is the head-to-tail fusion of acyl and malonyl building blocks, which leads to linear backbones. Yet, structural diversity is limited by this uniform assembly mode. Here we demonstrate a new type of PKS module from the endofungal bacterium Burkholderia rhizoxinica that catalyses a Michael-type acetyl addition to generate a branch in the carbon chain. In vitro reconstitution of the entire PKS module, X-ray structures of a ketosynthase-branching didomain and mutagenesis experiments revealed a crucial role of the ketosynthase domain in branching the carbon chain. We present a trapped intermediary state in which acyl carrier protein and ketosynthase are covalently linked by the branched polyketide and suggest a new mechanism for chain alkylation, which is functionally distinct from terpenoid-like β-branching. For the rice seedling blight toxin rhizoxin, one of the strongest known anti-mitotic agents, the non-canonical polyketide modification is indispensable for phytotoxic and anti-tumoral activities. We propose that the formation of related pharmacophoric groups follows the same general scheme and infer a unifying vinylogous branching reaction for PKS modules with a ketosynthase-branching–acyl-carrier-protein architecture. This study unveils the structure and function of a new PKS module that broadens the biosynthetic scope of polyketide biosynthesis and sets the stage for rationally creating structural diversity.

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Figure 1: Model of rhizoxin biosynthesis and chain branching mechanisms.
Figure 2: In vitro reconstitution of the chain branching reaction.
Figure 3: Domain structures of branching module (RhiE*).
Figure 4: Model of enzyme mechanism for Michael addition and lactone formation based on NMR and SDS–PAGE analyses.

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Protein Data Bank

Data deposits

The coordinates and structure factor amplitudes for RhiE* were deposited in the PDB database under accession code 4KC5.

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Acknowledgements

We thank A. Perner for mass specrometry analyses, H. Heineke for NMR measurements, S. Schneider for preliminary studies on the PPTase, and U. Knüpfer for help in protein production. We thank the DFG for financial support (SFB 766) and the Swiss Light Source beamline X06DA for offering beamtime. D.H. is financially supported by a stipend of the Studienstiftung des Deutschen Volkes.

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Authors and Affiliations

  1. Department of Biomolecular Chemistry, Leibniz Institute for Natural Product Research and Infection Biology (HKI), Jena 07745, Germany,

    Tom Bretschneider, Daniel Heine, Robert Winkler, Benjamin Busch, Björn Kusebauch & Christian Hertweck

  2. Interfaculty Institute of Biochemistry, Eberhard Karls University Tübingen, Tübingen 72076, Germany,

    Joel B. Heim, Thilo Stehle & Georg Zocher

  3. Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, 37232, Tennessee, USA

    Thilo Stehle

  4. Chair for Natural Product Chemistry, Friedrich Schiller University, Jena 07737, Germany,

    Christian Hertweck

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Contributions

T.B., G.Z. and C.H. designed experiments, T.B., R.W. and B.B. performed genetic and biochemical experiments and analysed data, D.H. and B.K. synthesized substrates and reference compounds, J.B.H., T.S. and G.Z. conducted protein crystallization, X-ray analyses and modelling, T.B., G.Z. and C.H. wrote the manuscript.

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Correspondence to Georg Zocher or Christian Hertweck.

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This file contains Supplementary Results and Discussion, Supplementary Materials and Methods, Supplementary Figures 1-13, Supplementary Tables 1-2 and additional references. (PDF 1590 kb)

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Bretschneider, T., Heim, J., Heine, D. et al. Vinylogous chain branching catalysed by a dedicated polyketide synthase module. Nature 502, 124–128 (2013). https://doi.org/10.1038/nature12588

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