TY - JOUR T1 - Structure-Function Analysis of Vitamin D 24-Hydroxylase (CYP24A1) by Site-Directed Mutagenesis: Amino Acid Residues Responsible for Species-Based Difference of CYP24A1 between Humans and Rats JF - Molecular Pharmacology JO - Mol Pharmacol SP - 120 LP - 128 DO - 10.1124/mol.106.023275 VL - 70 IS - 1 AU - Hiromi Hamamoto AU - Tatsuya Kusudo AU - Naoko Urushino AU - Hiroyuki Masuno AU - Keiko Yamamoto AU - Sachiko Yamada AU - Masaki Kamakura AU - Miho Ohta AU - Kuniyo Inouye AU - Toshiyuki Sakaki Y1 - 2006/07/01 UR - http://molpharm.aspetjournals.org/content/70/1/120.abstract N2 - Our previous studies revealed the species-based difference of CYP24A1-dependent vitamin D metabolism. Although human CYP24A1 catalyzes both C-23 and C-24 oxidation pathways, rat CYP24A1 shows almost no C-23 oxidation pathway. We tried to identify amino acid residues that cause the species-based difference by site-directed mutagenesis. In the putative substrate-binding regions, amino acid residue of rat CYP24A1 was converted to the corresponding residue of human CYP24A1. Among eight mutants examined, T416M and I500T showed C-23 oxidation pathway. In addition, the mutant I500F showed quite a different metabolism of 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] from both human and rat CYP24A1. These results strongly suggest 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)2D3 could be located close to amino acid residues at positions 416 and 500, respectively. Our findings provide useful information for the development of new vitamin D analogs for clinical use. ER -