Identification of the Amino Acid Residue of CYP27B1 Responsible for Binding of 25-Hydroxyvitamin D3 Whose Mutation Causes Vitamin D-dependent Rickets Type 1

We previously reported the three-dimensional structure of human CYP27B1 (25-hydroxyvitamin D₃ 1α-hydroxylase) constructed by homology modeling. Using the three-dimensional model we studied the docking of the substrate, 25-hydroxyvitamin D₃, into the substrate binding pocket of CYP27B1. In this study, we focused on the amino acid residues whose point mutations cause vitamin D-dependent rickets type 1, especially unconserved residues among mitochondrial CYPs such as Gln⁶⁵ and Thr⁴⁰⁹. Recently, we successfully overexpressed mouse CYP27B1 by using a GroEL/ES co-expression system. In a mutation study of mouse CYP27B1 that included spectroscopic analysis, we concluded that in a 1α-hydroxylation process, Ser⁴⁰⁸ of mouse CYP27B1 corresponding to Thr⁴⁰⁹ of human CYP27B1 forms a hydrogen bond with the 25-hydroxyl group of 25-hydroxyvitamin D₃. This is the first report that shows a critical amino acid residue recognizing the 25-hydroxyl group of the vitamin D₃.