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A Rieske oxygenase/epoxide hydrolase-catalysed reaction cascade creates oxygen heterocycles in mupirocin biosynthesis

Nature Catalysis volume 1, pages 968–976 (2018)Cite this article

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Abstract

Oxygen heterocycles—in particular, tetrahydropyrans (THPs) and tetrahydrofurans—are common structural features of many biologically active polyketide natural products. Mupirocin is a clinically important antibiotic isolated from Pseudomonas fluorescens and is assembled on a THP ring, which is essential for bioactivity. However, the biosynthesis of this moiety has remained elusive. Here, we show an oxidative enzyme-catalysed cascade that generates the THP ring of mupirocin. Rieske non-haem oxygenase (MupW)-catalysed selective oxidation of the C8–C16 single bond in a complex acyclic precursor is combined with an epoxide hydrolase (MupZ) to catalyse the subsequent regioselective ring formation to give the hydroxylated THP. In the absence of MupZ, a five-membered tetrahydrofuran ring is isolated, and model studies are consistent with cyclization occurring via an epoxide intermediate. High-resolution X-ray crystallographic studies, molecular modelling and mutagenesis experiments of MupZ provide insights into THP ring formation proceeding via an anti-Baldwin 6-endo-tet cyclization.

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Fig. 1: Summary of mupirocin biosynthesis in P. fluorescens NCIMB 10586.
Fig. 2: Characterization of the Rieske non-haem oxygenase MupW.
Fig. 3: Whole-cell biotransformations reveal MupW-catalysed oxidations.
Fig. 4: MupZ catalyses formation of the THP ring.
Fig. 5: X-ray structure of MupZ (PDB ID: 6FXD) and proposed mechanism of cyclization in mupirocin biosynthesis.
Fig. 6: Enzyme-catalysed reactions giving hydroxylated THPs via 6-endo-tet cyclizations.

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Data availability

The data that support the plots within this paper and other findings of this study are available from the corresponding authors upon reasonable request. X-ray crystallographic data are available in the EMBL-EBI PDB under accession number 6FXD.

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Acknowledgements

We are grateful to the BBSRC and EPSRC for funding through the Bristol Centre for Synthetic Biology (BB/L01386X/1), and the BBSRC for funding through BB/M012107/1 and BB/R007853/1, as well as a David Phillips Fellowship (BB/M026280/1) to M.W.v.d.K. We thank M. Malaysia for a scholarship to N.A.B., and H. P. Schweizer for kindly providing plasmids for gene inactivation and complementation.

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Authors and Affiliations

  1. School of Chemistry, University of Bristol, Bristol, UK

    Luoyi Wang, Christopher Williams, Nurfarhanim A. Bakar, Thomas J. Simpson, Matthew P. Crump & Christine L. Willis

  2. BrisSynBio Centre for Synthetic Biology Research, University of Bristol, Bristol, UK

    Luoyi Wang, Alice Parnell, Christopher Williams, Martin R. Challand, Marc W. van der Kamp, Thomas J. Simpson, Paul R. Race, Matthew P. Crump & Christine L. Willis

  3. School of Biochemistry, University of Bristol, Bristol, UK

    Alice Parnell, Martin R. Challand, Marc W. van der Kamp & Paul R. Race

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Contributions

L.W. and C.L.W. designed the experiments, analysed the data and, together with M.P.C. and T.J.S., drafted the manuscript. L.W. conducted the heterologous expression, protein purification, biotransformations, isolation and characterization of metabolites, and site-directed mutagenesis. A.P. and C.W. assisted with protein crystallization and structure determination. M.R.C. assisted with cloning and protein purification. N.A.B. conducted substrate synthesis and model cyclization. M.W.v.d.K. performed the molecular modelling. P.R.R. and M.P.C. led the protein structural studies and contributed to writing the manuscript.

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Correspondence to Matthew P. Crump or Christine L. Willis.

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Wang, L., Parnell, A., Williams, C. et al. A Rieske oxygenase/epoxide hydrolase-catalysed reaction cascade creates oxygen heterocycles in mupirocin biosynthesis. Nat Catal 1, 968–976 (2018). https://doi.org/10.1038/s41929-018-0183-5

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