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Tirandamycin biosynthesis is mediated by co-dependent oxidative enzymes

Nature Chemistry volume 3, pages 628–633 (2011)Cite this article

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Abstract

Elucidation of natural product biosynthetic pathways provides important insights into the assembly of potent bioactive molecules, and expands access to unique enzymes able to selectively modify complex substrates. Here, we show full reconstitution, in vitro, of an unusual multi-step oxidative cascade for post-assembly-line tailoring of tirandamycin antibiotics. This pathway involves a remarkably versatile and iterative cytochrome P450 monooxygenase (TamI) and a flavin adenine dinucleotide-dependent oxidase (TamL), which act co-dependently through the repeated exchange of substrates. TamI hydroxylates tirandamycin C (TirC) to generate tirandamycin E (TirE), a previously unidentified tirandamycin intermediate. TirE is subsequently oxidized by TamL, giving rise to the ketone of tirandamycin D (TirD), after which a unique exchange back to TamI enables successive epoxidation and hydroxylation to afford, respectively, the final products tirandamycin A (TirA) and tirandamycin B (TirB). Ligand-free, substrate- and product-bound crystal structures of bicovalently flavinylated TamL oxidase reveal a likely mechanism for the C10 oxidation of TirE.

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Figure 1: Tetramic acid natural products bearing a bicyclic ketal moiety (red) with varying degrees of oxidative modification.
Figure 2: Spectral analysis of tirandamycin tailoring enzymes.
Figure 3: Elucidation of individual steps in the tirandamycin oxidative cascade.
Figure 4: Ligand-free and substrate/product-bound TamL.

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Acknowledgements

The authors thank P. G. Cruz for assistance with NMR structure elucidation, P. M. Kells for excellent technical assistance, and staff members of Beamline 8.3.1, J. Holton, G. Meigs and J. Tanamachi at the Advanced Light Source at Lawrence Berkeley National Laboratory, for assistance with data collection. This work was funded by the National Institutes of Health (grant GM078553 to D.H.S. and L.M.P.), International Cooperative Biodiversity Group (U01 TW007404) and the Hans W. Vahlteich Professorship (to D.H.S.). The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences, of the US Department of Energy (contract no. DE-AC02-05CH11231).

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Author notes

  1. Jacob C. Carlson and Shengying Li: These authors contributed equally to this work

Authors and Affiliations

  1. Life Sciences Institute and Departments of Medicinal Chemistry, Microbiology and Immunology, and Chemistry, University of Michigan, Ann Arbor, 48109, Michigan, USA

    Jacob C. Carlson, Shengying Li, Yojiro Anzai, Douglas A. Burr & David H. Sherman

  2. Department of Pathology and Sandler Center for Drug Discovery, University of California, San Francisco, 94158, California, USA

    Shamila S. Gunatilleke & Larissa M. Podust

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Contributions

All authors designed experiments and analysed data. J.C., S.L., S.G., Y.A. and D.B. performed the experiments. J.C., S.L., L.P. and D.S. wrote the manuscript. J.C. and S.L. contributed equally to this study.

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Correspondence to David H. Sherman.

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The authors declare no competing financial interests.

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Carlson, J., Li, S., Gunatilleke, S. et al. Tirandamycin biosynthesis is mediated by co-dependent oxidative enzymes. Nature Chem 3, 628–633 (2011). https://doi.org/10.1038/nchem.1087

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