Phosphite-driven Self-sufficient Cytochrome P450

Authors

  • Hiroshi Watanabe,

    1. Department of Bioengineering, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan), Fax: (+81) 3-5841-8657
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  • Dr. Hidehiko Hirakawa,

    Corresponding author
    1. Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan)
    • Hidehiko Hirakawa, Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan)

      Teruyuki Nagamune, Department of Bioengineering, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan), Fax: (+81) 3-5841-8657

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  • Prof. Teruyuki Nagamune

    Corresponding author
    1. Department of Bioengineering, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan), Fax: (+81) 3-5841-8657
    2. Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan)
    • Hidehiko Hirakawa, Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan)

      Teruyuki Nagamune, Department of Bioengineering, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan), Fax: (+81) 3-5841-8657

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Abstract

Cytochrome P450s (P450s) are attractive for biotechnology applications because P450s can catalyze regio- and stereo-selective oxidations of non-activated hydrocarbons. Many bacterial P450s require electron transfer from ferredoxins, which are in turn reduced by specific ferredoxin reductases and NAD(P)H. Stoichiometric consumption of NADH, an expensive reagent, is a major challenge for practical applications. A phosphite dehydrogenase (PTDH) that catalyzes the oxidation of phosphite to phosphate using NAD+, is an attractive choice of enzyme for NADH regeneration. In this study, we incorporated PTDH into an artificial heterotrimeric complex of Pseudomonas putida P450, putidaredoxin and putidaredoxin reductase, and the resulting complex almost completely hydroxylated 1 mm d-camphor using 10 mM phosphite in the presence of 20 μM NAD+. This phosphite-driven self-sufficient P450 complex could provide a useful prototype for industry-scale oxidizing biocatalysts.

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