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A suicide enzyme catalyzes multiple reactions for biotin biosynthesis in cyanobacteria

Nature Chemical Biology volume 16, pages 415–422 (2020)Cite this article

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Abstract

In biotin biosynthesis, the conversion of pimeloyl intermediates to biotin is catalyzed by a universal set of four enzymes: BioF, BioA, BioD and BioB. We found that the gene homologous to bioA, the product of which is involved in the conversion of 8-amino-7-oxononanoate (AON) to 7,8-diaminononanoate (DAN), is missing in the genome of the cyanobacterium Synechocystis sp. PCC 6803. We provide structural and biochemical evidence showing that a novel dehydrogenase, BioU, is involved in biotin biosynthesis and functionally replaces BioA. This enzyme catalyzes three reactions: formation of covalent linkage with AON to yield a BioU-DAN conjugate at the ε-amino group of Lys124 of BioU using NAD(P)H, carboxylation of the conjugate to form BioU-DAN-carbamic acid, and release of DAN-carbamic acid using NAD(P)+. In this biosynthetic pathway, BioU is a suicide enzyme that loses the Lys124 amino group after a single round of reaction.

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Fig. 1: The biotin biosynthetic pathway and missing link in some haloarchaea and cyanobacteria.
Fig. 2: Phenotypic analysis of Synechocystis sp. PCC 6803-Δslr0355::Kmr and E. coli BW25113-ΔbioA.
Fig. 3: LC-HR-MS analysis for detection of the BioU-DAN conjugate and dethiobiotin.
Fig. 4: Proposed reaction of BioU via the BioU-DAN conjugate.
Fig. 5: Crystal structure of BioU.
Fig. 6: Proposed reaction mechanism of BioU.

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Data availability

Crystallographic data are available in the RCSB PDB with the identifiers 6IR4 for BioU in the resting state (P3121), 6K36 for the BioU-DAN conjugate (P3121), 6K38 for the BioU-H233A-DAN conjugate (P3121), 6ITD for AMENDA-bound BioU-K124A (P3121) and 6K37 for NAD+-bound BioU-K124A (P3121).

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Acknowledgements

This work was supported in part by JSPS KAKENHI (grant no. 17H06168 to M.N.) and the Research Committee of B group Vitamins (to M.N.). We are grateful to the staff at Photon Factory for their assistance with data collection, which was approved by the Photon Factory Program Advisory Committee (proposals nos. 2015G546, 2017G574 and 2019G548).

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Author notes

  1. These authors contributed equally: Kei Sakaki, Keita Ohishi.

Authors and Affiliations

  1. Biotechnology Research Center, The University of Tokyo, Bunkyo-ku, Tokyo, Japan

    Kei Sakaki, Keita Ohishi, Tetsu Shimizu, Kenichi Matsuda, Takeo Tomita, Tomohisa Kuzuyama & Makoto Nishiyama

  2. Laboratory for Chemistry and Life Science Institute of Innovative Research, Tokyo Institute of Technology, Midori-ku, Yokohama, Japan

    Ikki Kobayashi & Kan Tanaka

  3. Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan

    Naoki Mori & Hidenori Watanabe

  4. Collaborative Research Institute for Innovative Microbiology, The University of Tokyo, Bunkyo-ku, Tokyo, Japan

    Takeo Tomita, Tomohisa Kuzuyama & Makoto Nishiyama

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Contributions

K.S. and K.O performed cloning of bioU genes, biochemical analysis of BioU, crystallographic analysis of BioU and X-ray data correction. K.S. also wrote the manuscript. T.S. performed genome mining for bioU. I.K. and K.T. performed genetics analysis of bioU in Synechocystis sp. PCC 6803. H.W. and N.M. synthesized 7. K.M. analyzed the reaction intermediate. T.T. performed crystallographic analysis and structure refinements. T.K. and M.N. designed the project, analyzed the data and wrote the manuscript. All authors read and approved the final manuscript.

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Correspondence to Makoto Nishiyama.

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Supplementary Tables 1 and 2, Figs. 1–14 and Note.

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Sakaki, K., Ohishi, K., Shimizu, T. et al. A suicide enzyme catalyzes multiple reactions for biotin biosynthesis in cyanobacteria. Nat Chem Biol 16, 415–422 (2020). https://doi.org/10.1038/s41589-019-0461-9

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