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

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 V_max 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.     

Permeabilization induced by lipid II-targeting lantibiotic nisin and its effect on the bioconversion of vitamin D3 to 25-hydroxyvitamin D3 by Rhodococcus erythropolis

Vitamin D₃ (VD₃) is a fat-soluble prohormone in mammals. VD₃ is inert and must be activated by hydroxylation at the C-25 and C-1α positions to exert its biological activity. We recently accomplished the bioconversion of VD₃ to 25(OH)VD₃ with a recombinant strain of Rhodococcus erythropolis and found that the permeability of VD₃ into the cytoplasm may be the rate-limiting step of 25(OH)VD₃ production (Sallam et al., 2010). When the cells were treated with the lipid II-targeting lantibiotic nisin, the permeability of green chemiluminescent cyclodextrin (GCCD), which is used as a model substrate instead of VD₃-partially methylated-β-cyclodextrin (PMCD) complex, was drastically induced. Nisin also induced VD₃ hydroxylation, and the rate was correlated with the expression levels of Vdh and its redox partner proteins. In the bioconversion reaction, the stability of the redox partner proteins and the additional NADH-regenerating system are crucial for VD₃ hydroxylation. The degradation rate of the [2Fe–2S] cluster of ferredoxin ThcC from R. erythropolis NI86/21 is faster than that of AciB from Acinetobacter sp. OC4. Therefore, the nisin-treated R. erythropolis cells coexpressing Vdh and AciBC (1176.5 μg) exhibited much greater 25(OH)VD₃ production than the cells coexpressing Vdh and ThcCD (431.7 μg) after four consecutive 16 h reactions. These results suggest that nisin forms nisin-lipid II pore complexes in the Rhodococcus membrane that increase the accessibility of VD₃–PMCD complexes to the inside of the cells. Furthermore, nisin-treated Rhodococcus cells can be utilized for the bioconversion of other fat-soluble chemicals.