Abstract:

Vitamin D₃ (VD₃), a prohormone in mammals, plays a crucial role in the maintenance of calcium and phosphorus concentrations in serum. Activation of VD₃ requires 25-hydroxylation in the liver and 1α-hydroxylation in the kidney by cytochrome P450 (CYP) enzymes. Bacterial CYP105A1 converts VD₃ into 1α,25-dihydroxyvitamin D₃ (1α,25(OH)₂D₃) in two independent reactions, despite its low sequence identity with mammalian enzymes (<21% identity). The present study determined the crystal structures of a highly active mutant (R84A) of CYP105A1 from Streptomyces griseolus in complex and not in complex with 1α,25(OH)₂D₃. The compound 1α,25(OH)₂D₃ is positioned 11 Å from the iron atom along the I helix within the pocket. A similar binding mode is observed in the structure of the human CYP2R1−VD₃ complex, indicating a common substrate-binding mechanism for 25-hydroxylation. A comparison with the structure of wild-type CYP105A1 suggests that the loss of two hydrogen bonds in the R84A mutant increases the adaptability of the B′ and F helices, creating a transient binding site. Further mutational analysis of the active site reveals that 25- and 1α-hydroxylations share residues that participate in these reactions. These results provide the structural basis for understanding the mechanism of the two-step hydroxylation that activates VD₃.