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Reveromycin A biosynthesis uses RevG and RevJ for stereospecific spiroacetal formation

Nature Chemical Biology volume 7, pages 461–468 (2011)Cite this article

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Abstract

Spiroacetal compounds are ubiquitous in nature, and their stereospecific structures are responsible for diverse pharmaceutical activities. Elucidation of the biosynthetic mechanisms that are involved in spiroacetal formation will open the door to efficient generation of stereospecific structures that are otherwise hard to synthesize chemically. However, the biosynthesis of these compounds is poorly understood, owing to difficulties in identifying the responsible enzymes and analyzing unstable intermediates. Here we comprehensively describe the spiroacetal formation involved in the biosynthesis of reveromycin A, which inhibits bone resorption and bone metastases of tumor cells by inducing apoptosis in osteoclasts. We performed gene disruption, systematic metabolite analysis, feeding of labeled precursors and conversion studies with recombinant enzymes. We identified two key enzymes, dihydroxy ketone synthase and spiroacetal synthase, and showed in vitro reconstruction of the stereospecific spiroacetal structure from a stable acyclic precursor. Our findings provide insights into the creation of a variety of biologically active spiroacetal compounds for drug leads.

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Figure 1: Natural products containing a spiroacetal structure.
Figure 2: Deciphering of RM-PKS.
Figure 3: Post-PKS biosynthetic intermediate and mode of spiroacetal formation.
Figure 4: Analyses of enzyme reaction products.
Figure 5: Proposed biosynthetic pathway of 1. Brackets indicate the proposed dihydroxy ketone precursor (17) and the scheme for spiroacetal formation.

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Acknowledgements

We thank T. Shimizu for discussions on spiroacetal formation; K. Wierzba for reading this paper; T. Nakamura for mass analysis; N. Dohmae for MALDI-TOF and N-terminal sequencing; and E. Oowada for technical assistance. We thank all the technical staff of the Sequence Technology Team at RIKEN Genomic Sciences Center for their assistance. This work was supported in part by the Naito Foundation, the Strategic Programs for R&D of RIKEN, a Grant-in-Aid for Creative Scientific Research and a Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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Authors and Affiliations

  1. Chemical Biology Department, RIKEN Advanced Science Institute, Saitama, Japan

    Shunji Takahashi, Yasuyo Sekiyama, Hiroshi Takagi, Toshihiko Nogawa, Masakazu Uramoto, Ryuichiro Suzuki, Hiroyuki Koshino, Takuto Kumano, Suresh Panthee & Hiroyuki Osada

  2. Comparative Genomics Laboratory, National Institute of Genetics, Shizuoka, Japan

    Atsushi Toyoda

  3. Faculty of Engineering, Hokkaido University, Hokkaido, Japan

    Tohru Dairi

  4. Department of Bioactive Molecules, National Institute of Infectious Diseases, Tokyo, Japan

    Jun Ishikawa

  5. Kitasato Institute for Life Science, Kitasato University, Kanagawa, Japan

    Haruo Ikeda

  6. Toyohashi University of Technology, Aichi, Japan

    Yoshiyuki Sakaki

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Contributions

S.T.: the identification of RM gene cluster, genetic analysis, enzyme characterization and writing of the paper; A.T. and Y. Sakaki: sequencing of Streptomyces sp. SN-593 genome; Y. Sekiyama: the 13C-labeling experiments; H.T. and R.S.: the isolation of RM derivatives; T.N., M.U. and H.K.: the structural analyses; T.N. and M.U: the writing of structural analysis; S.P.: the analysis of the border genes; T.K.: the characterization of RevJ; T.D.: the RT-PCR analysis; J.I.: the development of the gene annotation system; H.I.: discussion about the gene transformation system for Streptomyces sp. SN-593; H.O.: the integration of all research. All authors discussed the results and commented on the manuscript.

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Correspondence to Hiroyuki Osada.

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Takahashi, S., Toyoda, A., Sekiyama, Y. et al. Reveromycin A biosynthesis uses RevG and RevJ for stereospecific spiroacetal formation. Nat Chem Biol 7, 461–468 (2011). https://doi.org/10.1038/nchembio.583

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