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

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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.

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.

References

  1. 1.

    Newman, D. J. & Cragg, G. M. Natural products as sources of new drugs from 1981 to 2014. J. Nat. Prod. 79, 629–661 (2016).

    CAS  Article  PubMed  Google Scholar 

  2. 2.

    Carlson, J. C. et al. Tirandamycin biosynthesis is mediated by co-dependent oxidative enzymes. Nat. Chem. 3, 628–633 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Bridwell-Rabb, J., Kang, G., Zhong, A., Liu, H. W. & Drennan, C. L. An HD domain phosphohydrolase active site tailored for oxetanocin-A biosynthesis. Proc. Natl Acad. Sci. USA 113, 13750–13755 (2016).

    CAS  Article  PubMed  Google Scholar 

  4. 4.

    Fuller, A. T. et al. Pseudomonic acid: an antibiotic produced by Pseudomonas fluorescens. Nature 234, 416–417 (1971).

    CAS  Article  PubMed  Google Scholar 

  5. 5.

    Thomas, C. M., Hothersall, J., Willis, C. L. & Simpson, T. J. Resistance to and synthesis of the antibiotic mupirocin. Nat. Rev. Microbiol. 8, 281–289 (2010).

    CAS  Article  PubMed  Google Scholar 

  6. 6.

    Martin, F. M. & Simpson, T. J. Biosynthetic studies on pseudomonic acid (mupirocin), a novel antibiotic metabolite of Pseudomonas fluorescens. J. Chem. Soc. Perkin Trans. 1, 207–209 (1989).

    Article  Google Scholar 

  7. 7.

    EI-Sayed, A. K. et al. Characterization of the mupirocin biosynthesis gene cluster from Pseudomonas fluorescens NCIMB 10586. Chem. Biol. 10, 419–430 (2003).

    Article  Google Scholar 

  8. 8.

    Hothersall, J. et al. Mutational analysis reveals that all tailoring region genes are required for production of polyketide antibiotic mupirocin by Pseudomonas fluorescens: pseudomonic acid B biosynthesis precedes pseudomonic acid A. J. Biol. Chem. 282, 15451–15461 (2007).

    CAS  Article  PubMed  Google Scholar 

  9. 9.

    Gao, S.-S. et al. Biosynthesis of mupirocin by Pseudomonas fluorescens NCIMB 10586 involves parallel pathways. J. Am. Chem. Soc. 136, 5501–5507 (2014).

    CAS  Article  PubMed  Google Scholar 

  10. 10.

    Gao, S.-S. et al. Selected mutations reveal new intermediates in the biosynthesis of mupirocin and the thiomarinol antibiotics. Angew. Chem. Int. Ed. 56, 3930–3934 (2017).

    CAS  Article  Google Scholar 

  11. 11.

    Silvian, L. F., Wang, J. & Steitz, T. A. Insights into editing from an Ile-tRNA synthetase structure with tRNAIle and mupirocin. Science 285, 1074–1077 (1999).

    CAS  Article  PubMed  Google Scholar 

  12. 12.

    Hemmerling, F. & Hahn, F. Biosynthesis of oxygen and nitrogen-containing heterocycles in polyketides. Beilstein J. Org. Chem. 12, 1512–1550 (2016).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Taylor, R. D., MacCoss, M. & Lawson, A. D. G. Rings in drugs. J. Med. Chem. 57, 5845–5859 (2014).

    CAS  Article  PubMed  Google Scholar 

  14. 14.

    Luhavaya, H. et al. Enzymology of pyran ring A formation in salinomycin biosynthesis. Angew. Chem. Int. Ed. 54, 13622–13625 (2015).

    CAS  Article  Google Scholar 

  15. 15.

    Cooper, S. M. et al. Mupirocin W, a novel pseudomonic acid produced by targeted mutation of the mupirocin biosynthetic gene cluster. Chem. Commun. 9, 1179–1181 (2005).

    Article  CAS  Google Scholar 

  16. 16.

    Kauppi, B. et al. Structure of an aromatic-ring-hydroxylating dioxygenase-naphthalene 1,2-dioxygenase. Structure 6, 571–586 (1998).

    CAS  Article  PubMed  Google Scholar 

  17. 17.

    Sydor, P. K. et al. Regio- and stereodivergent antibiotic oxidative carbocyclizations catalysed by Rieske oxygenase-like enzymes. Nat. Chem. 3, 388–392 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Suen, W. C. & Gibson, D. T. Isolation and preliminary characterization of the subunits of the terminal component of naphthalene dioxygenase from Pseudomonas putida NCIB 9816-4. J. Bacteriol. 175, 5877–5881 (1993).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Ferraro, D. J., Gakhar, L. & Ramaswamy, S. Rieske business: structure-function of Rieske non-heme oxygenases. Biochem. Biophys. Res. Commun. 338, 175–190 (2005).

    CAS  Article  PubMed  Google Scholar 

  20. 20.

    Li, B. et al. Whole-cell biotransformation systems for reduction of prochiral carbonyl compounds to chiral alcohol in Escherichia coli. Sci. Rep. 4, 6750 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Gally, C., Nestl, B. M. & Hauer, B. Engineering Rieske non-heme iron oxygenases for the asymmetric dihydroxylation of alkenes. Angew. Chem. Int. Ed. 54, 12952–12956 (2015).

    CAS  Article  Google Scholar 

  22. 22.

    Jouanneau, Y., Meyer, C. & Duraffourg, N. Dihydroxylation of four- and five-ring aromatic hydrocarbons by the naphthalene dioxygenase from Sphingomonas CHY-1. Appl. Microbiol. Biotechnol. 100, 1253–1263 (2016).

    CAS  Article  PubMed  Google Scholar 

  23. 23.

    Kan, S. B. J., Huang, X., Gumulya, Y., Chen, K. & Arnold, F. H. Genetically programmed chiral organoborane synthesis. Nature 552, 132–136 (2017).

    CAS  PubMed  Google Scholar 

  24. 24.

    Baldwin, J. E. Rules for ring closure. J. Chem. Soc. Chem. Comm. 734–736 (1976).

  25. 25.

    Gilmore, K. & Alabugin, I. V. Cyclizations of alkynes: revisiting Baldwin’s rules for ring closure. Chem. Rev. 111, 6513–6556 (2011).

    CAS  Article  PubMed  Google Scholar 

  26. 26.

    Gilmore, K., Mohamed, R. K. & Alabugin, I. V. The Baldwin rules: revised and extended. WIREs Comput. Mol. Sci. 6, 487–514 (2016).

    CAS  Article  Google Scholar 

  27. 27.

    Buchan, D. W., Minneci, F., Nugent, T. C., Bryson, K. & Jones, D. T. Scalable web services for the PSIPRED Protein Analysis Workbench. Nucleic Acids Res. 41, W349–W357 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Hmelo, L. R. et al. Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange. Nat. Protoc. 10, 1820–1841 (2015).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Holm, L. & Laakso, L. M. Dali server update. Nucleic Acids Res. 44, W351–W355 (2016).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Hotta, K. et al. Enzymatic catalysis of anti-Baldwin ring closure in polyether biosynthesis. Nature 483, 355–358 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Minami, A. et al. Allosteric regulation of epoxide opening cascades by a pair of epoxide hydrolases in monensin biosynthesis. ACS Chem. Biol. 9, 562–569 (2014).

    CAS  Article  PubMed  Google Scholar 

  32. 32.

    Capyk, J. K., D’Angelo, I., Strynadka, N. C. & Eltis, L. D. Characterization of 3-ketosteroid 9α-hydroxylase, a Rieske oxygenase in the cholesterol degradation pathway of Mycobacterium tuberculosis. J. Biol. Chem. 284, 9937–9946 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Yoshiyama-Yanagawa, T. et al. The conserved Rieske oxygenase DAF-36/Neverland is a novel cholesterol-metabolizing enzyme. J. Biol. Chem. 286, 25756–25762 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Li., J., van Belkum, M. J. & Vederas, J. C. Functional characterization of recombinant hyoscyamine 6β-hydroxylase from Atropa belladonna. Bioorg. Med. Chem. 20, 4356–4363 (2012).

    CAS  Article  PubMed  Google Scholar 

  35. 35.

    Mao, X.-M. et al. Efficient biosynthesis of fungal polyketides containing the dioxabicyclo-octane ring system. J. Am. Chem. Soc. 137, 11904–11907 (2015).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Minami, A. et al. Sequential enzymatic epoxidation involved in polyether lasalocid biosynthesis. J. Am. Chem. Soc. 134, 7246–7249 (2012).

    CAS  Article  PubMed  Google Scholar 

  37. 37.

    Shichijo, Y. et al. Epoxide hydrolase Lsd19 for polyether formation in the biosynthesis of lasalocid A: direct experimental evidence on polyene-polyepoxide hypothesis in polyether biosynthesis. J. Am. Chem. Soc. 130, 12230–12231 (2008).

    CAS  Article  PubMed  Google Scholar 

  38. 38.

    Barry, S. M. & Challis, G. L. Mechanism and catalytic diversity of Rieske non-heme iron-dependent oxygenases. ACS Catal. 3, 2362–2370 (2013).

    CAS  Article  Google Scholar 

  39. 39.

    Parkhurst, J. M. et al. Dials. J. Appl. Crystallogr. 49, 1912–1921 (2016).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Winter, G. xia2: an expert system for macromolecular crystallography data reduction. J. Appl. Crystallogr. 43, 186–190 (2010).

    CAS  Article  Google Scholar 

  41. 41.

    Vagin, A. & Teplyakov, A. Molecular replacement with MOLREP. Acta Crystallogr. D Biol. Crystallogr. 66, 22–25 (2010).

    CAS  Article  PubMed  Google Scholar 

  42. 42.

    Potterton, L. et al. CCP4i2: the new graphical user interface to the CCP4 program suite. Acta Crystallogr. D Struct. Biol. 74, 68–84 (2018).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Cowtan, K. The Buccaneer software for automated model building. 1. Tracing protein chains. Acta Crystallogr. D Struct. Biol. 62, 1002–1011 (2006).

    Article  CAS  Google Scholar 

  45. 45.

    Emsley, P., Lohkamp, B., Scottc, W. G. & Cowtand, K. Features and development of Coot. Acta Crystallogr. D Struct. Biol. 66, 486–501 (2010).

    CAS  Article  Google Scholar 

  46. 46.

    Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D53, 240–255 (1997).

    CAS  Google Scholar 

  47. 47.

    Frisch, M. J. et al. Gaussian (Wallingford, 2009).

  48. 48.

    Trott, O. & Olson, A. J. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading. J. Comput. Chem. 31, 455–461 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. 49.

    Case, D. A. et al. AMBER 2016 (Univ. California, 2016).

  50. 50.

    Sondergaard, C. R., Olsson, M. H. M., Rostkowski, M. & Jensen, J. H. Improved treatment of ligands and coupling effects in empirical calculation and rationalization of pKa values. J. Chem. Theory Comput. 7, 2284–2295 (2011).

    CAS  Article  PubMed  Google Scholar 

  51. 51.

    Olsson, M. H. M., Sondergaard, C. R., Rostkowski, M. & Jensen, J. H. PROPKA3: consistent treatment of internal and surface residues in empirical pKa predictions. J. Chem. Theory Comput. 7, 525–537 (2011).

    CAS  Article  PubMed  Google Scholar 

  52. 52.

    Maier, J. A. et al. ff14SB: improving the accuracy of protein side chain and backbone parameters from ff99SB. J. Chem. Theory Comput. 11, 3696–3713 (2015).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  53. 53.

    Wang, J., Wolf, R. M., Caldwell, J. W., Kollman, P. A. & Case, D. A. Development and testing of a general amber force field. J. Comput. Chem. 25, 1157–1174 (2004).

    CAS  Article  PubMed  Google Scholar 

Download references

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.

Author information

Affiliations

Authors

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.

Corresponding authors

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

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Methods, Supplementary Figures 1–48, Supplementary Tables 1–2

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

Further reading

Search

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing