Purification, characterization, and directed evolution study of a vitamin D3 hydroxylase from Pseudonocardia autotrophica

Yoshikazu Fujii, Hiroki Kabumoto, Kenji Nishimura, Tadashi Fujii, Satoshi Yanai, Koji Takeda, Noriko Tamura, Akira Arisawa, Tomohiro Tamura

Research output: Contribution to journalArticlepeer-review

59 Citations (Scopus)

Abstract

Vitamin D3 (VD3) is a fat-soluble prohormone that plays a crucial role in bone metabolism, immunity, and control of cell proliferation and cell differentiation in mammals. The actinomycete Pseudonocardia autotrophica is capable of bioconversion of VD3 into its physiologically active forms, namely, 25(OH)VD3 or 1α,25(OH)2VD3. In this study, we isolated and characterized Vdh (vitamin D3 hydroxylase), which hydroxylates VD3 from P. autotrophica NBRC 12743. The vdh gene encodes a protein containing 403 amino acids with a molecular weight of 44,368 Da. This hydroxylase was found to be homologous with the P450 belonging to CYP107 family. Vdh had the same ratio of the Vmax values for VD3 25-hydroxylation and 25(OH)VD3 1α-hydroxylation, while other enzymes showed preferential regio-specific hydroxylation on VD3. We characterized a collection of Vdh mutants obtained by random mutagenesis and obtained a Vdh-K1 mutant by the combination of four amino acid substitutions. Vdh-K1 showed one-order higher VD3 25-hydroxylase activity than the wild-type enzyme. Biotransformation of VD3 into 25(OH)VD3 was successfully accomplished with a Vdh-expressed recombinant strain of actinobacterium Rhodococcus erythropolis. Vdh may be a useful enzyme for the production of physiologically active forms of VD3 by a single cytochrome P450.

Original languageEnglish
Pages (from-to)170-175
Number of pages6
JournalBiochemical and Biophysical Research Communications
Volume385
Issue number2
DOIs
Publication statusPublished - 24-07-2009
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint

Dive into the research topics of 'Purification, characterization, and directed evolution study of a vitamin D3 hydroxylase from Pseudonocardia autotrophica'. Together they form a unique fingerprint.

Cite this