title: Purification, characterization, and directed evolution study of a vitamin D3 hydroxylase from Pseudonocardia autotrophica.
abstract: Vitamin D(3) (VD(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(3) into its physiologically active forms, namely, 25(OH)VD(3) or 1alpha,25(OH)(2)VD(3). In this study, we isolated and characterized Vdh (vitamin D(3) hydroxylase), which hydroxylates VD(3) from P. autotrophica NBRC 12743. The vdh gene encodes a protein containing 403 amino acids with a molecular weight of 44,368Da. This hydroxylase was found to be homologous with the P450 belonging to CYP107 family. Vdh had the same ratio of the V(max) values for VD(3) 25-hydroxylation and 25(OH)VD(3) 1alpha-hydroxylation, while other enzymes showed preferential regio-specific hydroxylation on VD(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(3) 25-hydroxylase activity than the wild-type enzyme. Biotransformation of VD(3) into 25(OH)VD(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(3) by a single cytochrome P450.
authors.affiliation: Bioresource Laboratories, Mercian Corporation, Iwata, Shizuoka, Japan.
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Fujii
authors.forename: Hiroki
authors.initials: H
authors.lastname: Kabumoto
authors.forename: Kenji
authors.initials: K
authors.lastname: Nishimura
authors.forename: Tadashi
authors.initials: T
authors.lastname: Fujii
authors.forename: Satoshi
authors.initials: S
authors.lastname: Yanai
authors.forename: Koji
authors.initials: K
authors.lastname: Takeda
authors.forename: Noriko
authors.initials: N
authors.lastname: Tamura
authors.forename: Akira
authors.initials: A
authors.lastname: Arisawa
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Tamura
mesh_terms: D000192:Actinomycetales / Q000201:enzymology*; D000595:Amino Acid Sequence; D053493:Cholestanetriol 26-Monooxygenase / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D019020:Directed Molecular Evolution; D008969:Molecular Sequence Data
chemical_list: D053493:Cholestanetriol 26-Monooxygenase