Purification, characterization, and directed evolution study of a vitamin D3 hydroxylase from Pseudonocardia autotrophica
| Autor: | Tadashi Fujii, Yoshikazu Fujii, Hiroki Kabumoto, Kenji Nishimura, Noriko Tamura, Tomohiro Tamura, Akira Arisawa, Koji Takeda, Satoshi Yanai |
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| Rok vydání: | 2009 |
| Předmět: |
chemistry.chemical_classification
biology Stereochemistry Molecular Sequence Data Mutant Biophysics Cytochrome P450 Cell Biology biology.organism_classification Biochemistry Cofactor Amino acid Hydroxylation chemistry.chemical_compound Enzyme chemistry Actinomycetales biology.protein Cholestanetriol 26-Monooxygenase Production (computer science) Amino Acid Sequence Cloning Molecular Directed Molecular Evolution Molecular Biology Rhodococcus |
| Zdroj: | Biochemical and Biophysical Research Communications. 385:170-175 |
| ISSN: | 0006-291X |
| DOI: | 10.1016/j.bbrc.2009.05.033 |
| Popis: | 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 ofmore » VD{sub 3} by a single cytochrome P450.« less |
| Databáze: | OpenAIRE |
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