Journal of Biological Chemistry p. 31193 - 31201 (2010)
Update date:2022-08-28
Topics:
Yasutake, Yoshiaki
Fujii, Yoshikazu
Nishioka, Taiki
Cheon, Woo-Kwang
Arisawa, Akira
Tamura, Tomohiro
Vitamin D3 hydroxylase (Vdh) isolated from actinomycete Pseudonocardia autotrophica is a cytochrome P450 (CYP) responsible for the biocatalytic conversion of vitamin D3 (VD3) to 1α,25-dihydroxyvitamin D3 (1α,25(OH)2VD 3) by P. autotrophica. Although its biological function is unclear, Vdh is capable of catalyzing the two-step hydroxylation of VD3, i.e. the conversion of VD3 to 25-hydroxyvitamin D3 (25(OH)VD3) and then of 25(OH)VD3 to 1α,25(OH) 2VD3, a hormonal form of VD3. Here we describe the crystal structures of wild-type Vdh (Vdh-WT) in the substrate-free form and of the highly active quadruple mutant (Vdh-K1) generated by directed evolution in the substrate-free, VD3-bound, and 25(OH)VD3-bound forms. Vdh-WT exhibits an open conformation with the distal heme pocket exposed to the solvent both in the presence and absence of a substrate, whereas Vdh-K1 exhibits a closed conformation in both the substrate-free and substrate-bound forms. The results suggest that the conformational equilibrium was largely shifted toward the closed conformation by four amino acid substitutions scattered throughout the molecule. The substratebound structure of Vdh-K1 accommodates both VD3 and 25(OH)VD3 but in an anti-parallel orientation. The occurrence of the two secosteroid binding modes accounts for the regioselective sequential VD3 hydroxylation activities. Moreover, these structures determined before and after directed evolution, together with biochemical and spectroscopic data, provide insights into how directed evolution has worked for significant enhancement of both the VD3 25-hydroxylase and 25(OH)VD3 1α-hydroxylase activities.
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