|Budget Amount *help
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2002: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2001: ¥2,600,000 (Direct Cost: ¥2,600,000)
We have determined eight types of missense mutants of CYP27B1 from vitamin D-dependent rickets type I patients, and examined their enzymatic properties. In addition, we generated various mutants of CYP27B1 and CYP27A1 to examine the roles of the mutated amino acid residues of the CYP27B1 mutants. The experimental data strongly suggested that they were involved in supply of protons, heme-propionate binding, activation of oxygen, substrate-binding, and stabilization of tertiary structure. Based on these results, the tertiary structures of CYP27B1 and CYP27A1 were constructed by computer modeling.
On CYP24-dependent metabolism of three vitamin D analogs described below, clear species-based difference was observed between humans and rats. The recombinant system harboring human CYP24 appears quite useful for predicting the metabolism and efficacy of vitamin D analogs in human target tissues before clinical trials. When F_6-1α,25(OH)_2D_3 was added to the recombinant E. coli cells expressing human CYP24, the putative ether compound with no affinity for vitamin D receptor (VDR) was observed. However, this metabolite was not observed in those expressing rat CYP24. On the metabolism of maxacalcitol, human CYP24 cleaved the O_<22>-C_<23> bond at the first step of the metabolism, while rat CYP24 cleaved it at the third step, suggesting rapid inactivation of maxacalcitol in human body. In the case of the metabolism of 1α,25(OH)_2-20-epi-D_3, rat CYP24 converted 1α,25(OH)_2-20-epi-D_3 to 25.26,27-trinor-lα (OH)-24(COOH) indicating completely inactivation of 1α,25(OH)_2-20-epi-D_3. On the other hand, human CYP24 mainly converted 1α,25(OH)_2-20-epi-D_3 to 1α,23,25(OH)_3-24-oxo-20-epi-D_3 which retains the affinity for VDR.