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Distinct mechanisms for spiro-carbon formation reveal biosynthetic pathway crosstalk

Nature Chemical Biology volume 9, pages 818–825 (2013)Cite this article

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Abstract

Spirotryprostatins, an indole alkaloid class of nonribosomal peptides isolated from Aspergillus fumigatus, are known for their antimitotic activity in tumor cells. Because spirotryprostatins and many other chemically complex spiro-carbon–bearing natural products exhibit useful biological activities, identifying and understanding the mechanism of spiro-carbon biosynthesis is of great interest. Here we report a detailed study of spiro-ring formation in spirotryprostatins from tryprostatins derived from the fumitremorgin biosynthetic pathway, using reactants and products prepared with engineered yeast and fungal strains. Unexpectedly, FqzB, an FAD-dependent monooxygenase from the unrelated fumiquinazoline biosynthetic pathway, catalyzed spiro-carbon formation in spirotryprostatin A via an epoxidation route. Furthermore, FtmG, a cytochrome P450 from the fumitremorgin biosynthetic pathway, was determined to catalyze the spiro-ring formation in spirotryprostatin B. Our results highlight the versatile role of oxygenating enzymes in the biosynthesis of structurally complex natural products and indicate that cross-talk of different biosynthetic pathways allows product diversification in natural product biosynthesis.

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Figure 1: Proposed biosynthesis of tryprostatins fumitremorgins and spirotryprostatins in A. fumigatus.
Figure 2: Proposed mechanisms of chemical transformations facilitated by epoxidation.
Figure 3: In vivo and in vitro characterization of the flavin-containing monooxygenase FqzB from the fumiquinazoline biosynthetic pathway in the formation of spirotryprostatin A.
Figure 4: In vivo and in vitro characterizations of the cytochrome P450 FtmG for the formation of spirotryprostatins B and G.

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Acknowledgements

We would like to thank J. Shimokawa at the University of Tokyo for providing us with a sample of spirotryprostatin A. We would like to express our appreciation to financial support from Japan Society for the Promotion of Science (JSPS) through the 'Funding Program for Next Generation World-Leading Researchers', initiated by the Council for Science and Technology Policy (no. LS103) (K.W.) and by the Industrial Technology Research Grant Program in 2009 (no. 09C46001a) from the New Energy and Industrial Technology Development Organization (NEDO) of Japan (K.W.). These works were also supported in part by The Uehara Memorial Foundation (K.W.), by Mochida Memorial Foundation for Medical and Pharmaceutical Research (K.W.), by The Hokuto Foundation for Bioscience (K.W.) and by The Naito Foundation Japan (K.W.). Postdoctoral fellowships to Y.T. from JSPS are gratefully acknowledged.

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Authors and Affiliations

  1. Department of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan

    Yuta Tsunematsu, Noriyasu Ishikawa, Hiroshi Noguchi & Kenji Watanabe

  2. National Institute of Health Sciences, Tokyo, Japan

    Daigo Wakana & Yukihiro Goda

  3. Research Core for Interdisciplinary Sciences, Okayama University, Okayama, Japan

    Hisao Moriya

  4. School of Biosciences, The University of Nottingham Malaysia Campus, Selangor, Malaysia

    Kinya Hotta

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  1. Yuta Tsunematsu
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Contributions

Y.T., N.I., H.M. and K.W. conceived and designed the study. Y.T. and N.I. designed and performed molecular cloning. Y.T. and N.I. performed the heterologous protein expression and purification as well as in vitro and in vivo characterization of the enzymes. D. W. and Y. G. elucidated the chemical structures. All of the authors analyzed and discussed the results. K.H. and K.W. prepared the manuscript.

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Correspondence to Kenji Watanabe.

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Supplementary Results, Supplementary Figures 1–68, Supplementary Tables 1–14 and Supplementary Notes 1–3. (PDF 7469 kb)

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Tsunematsu, Y., Ishikawa, N., Wakana, D. et al. Distinct mechanisms for spiro-carbon formation reveal biosynthetic pathway crosstalk. Nat Chem Biol 9, 818–825 (2013). https://doi.org/10.1038/nchembio.1366

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