Purification, characterization, and directed evolution study of a vitamin D{sub 3} hydroxylase from Pseudonocardia autotrophica

Journal Article · Fri Jul 24 00:00:00 EDT 2009 · Biochemical and Biophysical Research Communications

DOI:https://doi.org/10.1016/J.BBRC.2009.05.033· OSTI ID:22199743

Fujii, Yoshikazu ^[1]; Kabumoto, Hiroki; Nishimura, Kenji; Fujii, Tadashi; Yanai, Satoshi ^[1]; Takeda, Koji ^[2]; Tamura, Noriko ^[3]; Arisawa, Akira ^[1]; Tamura, Tomohiro ^[3]

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1. Bioresource Laboratories, Mercian Corporation, 1808 Nakaizumi, Iwata, Shizuoka 438-0078 (Japan)
2. BioTechnical Development Center, Mercian Corporation, 1808 Nakaizumi, Iwata, Shizuoka 438-0078 (Japan)
3. Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, Toyohira-ku, Sapporo 062-8517 (Japan)

Vitamin D{sub 3} (VD{sub 3}) 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 VD{sub 3} into its physiologically active forms, namely, 25(OH)VD{sub 3} or 1{alpha},25(OH){sub 2}VD{sub 3}. In this study, we isolated and characterized Vdh (vitamin D{sub 3} hydroxylase), which hydroxylates VD{sub 3} 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 V{sub max} values for VD{sub 3} 25-hydroxylation and 25(OH)VD{sub 3} 1{alpha}-hydroxylation, while other enzymes showed preferential regio-specific hydroxylation on VD{sub 3}. 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 VD{sub 3} 25-hydroxylase activity than the wild-type enzyme. Biotransformation of VD{sub 3} into 25(OH)VD{sub 3} 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 VD{sub 3} by a single cytochrome P450.

• OSTI ID: 22199743
• Journal Information: Biochemical and Biophysical Research Communications, Vol. 385, Issue 2; Other Information: Copyright (c) 2009 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA); ISSN 0006-291X
• Country of Publication: United States
• Language: English

Related Subjects

60 APPLIED LIFE SCIENCES
AMINO ACIDS
BIOCONVERSION
CELL DIFFERENTIATION
CELL PROLIFERATION
FERREDOXIN
GELS
HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY
HYDROXYLASES
HYDROXYLATION
IMMUNITY
MAMMALS
METABOLISM
MOLECULAR WEIGHT
MUTAGENESIS
MUTANTS
NADP
OLIGOSACCHARIDES
RHODOCOCCUS
SKELETON
SODIUM
SULFATES
VITAMIN D