OKANO Toshio Kobe Pharmaceutical Univ, Dept.Hygienic Sciences, Professor, 衛生化学研究室, 教授 (20131542)
TAKITA Teisuke Kyoto Univ, Dept.Enzyme Chemistry, Assistant Professor, 大学院・農学研究科, 助手 (70263126)
|Budget Amount *help
¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 2005: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 2004: ¥2,200,000 (Direct Cost: ¥2,200,000)
Mouse CYP27B1 was successfully overexpressed 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 D_3. In addition, we revealed that Arg458 of mouse CYP27B1 is responsible for the interaction with adrenodoxin.
We revealed the species-based difference of CYP24A-dependent vitamin D metabolism, and tried to identify amino-acid residues that cause the species-based difference by site-directed mutagenesis. It was demonstrated that the amino-acid residues at positions 416 and 500 play a crucial role in substrate-binding, and greatly affect substrate orientation. A three-dimensional model of CYP24A1 indicated that the A-ring and triene part of 1α,25(OH)_2D_3 could be located close to amino acid residues at positions 416 and 500, respectively.
We examined CYP24A1-dependent metabolism of such vitamin D analogs as A-ring diastereomers and 20-epimer of 1α,25(OH)_2D_3, 2α-propoxy- 1α,25(OH)_2D_3 2α-(3-hydroxypropoxy)- 1α,25(OH)_2D_3, and 26,26,26,27,27,27- F_6-1α,25(OH)_2D_3. Good correlation was observed between increasing effect on serum calcium level in rats and k_<cat>/K_m value of CYP24A1 for each of the vitamin D analogs. In addition, species-based difference was also observed in CYP24A1-dependent metabolism of vitamin D analogs between humans and rats. These results strongly suggest the usefulness of the recombinant systems harboring CYP24A1 for predicting the metabolism and efficacy of vitamin D analogs before clinical trials.