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A Single Mutation at the Ferredoxin Binding Site of P450 Vdh Enables Efficient Biocatalytic Production of 25-Hydroxyvitamin D3

Authors

  • Dr. Yoshiaki Yasutake,

    1. Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1, Tsukisamu-higashi, Toyohira-ku, Sapporo 062-8517 (Japan)
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    • These authors contributed equally to this work.

  • Dr. Taiki Nishioka,

    1. Graduate School of Agriculture, Hokkaido University, Kita-9, Nishi-9, Kita-ku, Sapporo 060-8589 (Japan)
    2. Current address: MicroBiopharm Japan, Co. Ltd. 1808 Nakaizumi, Iwata, Shizuoka 438-0078 (Japan)
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    • These authors contributed equally to this work.

  • Dr. Noriko Imoto,

    1. Graduate School of Agriculture, Hokkaido University, Kita-9, Nishi-9, Kita-ku, Sapporo 060-8589 (Japan)
    2. Current address: Department of Human Health Science,Hachinohe Gakuin University, 13–98 Mihono, Hachinohe, Aomori 031-8588 (Japan)
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  • Prof. Tomohiro Tamura

    Corresponding author
    1. Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1, Tsukisamu-higashi, Toyohira-ku, Sapporo 062-8517 (Japan)
    2. Graduate School of Agriculture, Hokkaido University, Kita-9, Nishi-9, Kita-ku, Sapporo 060-8589 (Japan)
    • Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1, Tsukisamu-higashi, Toyohira-ku, Sapporo 062-8517 (Japan)

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Abstract

Vitamin D3 hydroxylase (Vdh) from Pseudonocardia autotrophica is a cytochrome P450 monooxygenase that catalyzes the two-step hydroxylation of vitamin D3 (VD3) to produce 25-hydroxyvitamin D3 (25(OH)VD3) and 1α,25-dihydroxyvitamin D3 (1α,25(OH)2VD3). These hydroxylated forms of VD3 are useful as pharmaceuticals for the treatment of conditions associated with VD3 deficiency and VD3 metabolic disorder. Herein, we describe the creation of a highly active T107A mutant of Vdh by engineering the putative ferredoxin-binding site. Crystallographic and kinetic analyses indicate that the T107A mutation results in conformational change from an open to a closed state, thereby increasing the binding affinity with ferredoxin. We also report the efficient biocatalytic synthesis of 25(OH)VD3, a promising intermediate for the synthesis of various hydroxylated VD3 derivatives, by using nisin-treated Rhodococcus erythropolis cells containing VdhT107A. The gene-expression cassette encoding Bacillus megaterium glucose dehydrogenase-IV was inserted into the R. erythropolis chromosome and expressed to avoid exhaustion of NADH in a cytoplasm during bioconversion. As a result, approximately 573 μg mL−1 25(OH)VD3 was successfully produced by a 2 h bioconversion.

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