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First published on April 14, 2006; DOI: 10.1124/mol.106.023275

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Received for publication February 7, 2006.
Revised April 13, 2006.
Accepted for publication April 13, 2006.

Structure-function analysis of vitamin D 24-hydroxylase (CYP24A1) by site-directed mutagenesis: amino acid resides responsible for species-based difference of CYP24A1 between humans and rats

Hiromi Hamamoto 1, Tatsuya Kusudo 1, Naoko Urushino 1, Hiroyuki Masuno 2, Keiko Yamamoto 2, Sachiko Yamada 2, Masaki Kamakura 3, Miho Ohta 4, Kuniyo Inouye 1, Toshiyuki Sakaki 3*

1 Kyoto University 2 Tokyo Medical and Dental University 3 Toyama Prefectural University 4 Koshien College

* Address correspondence to: E-mail: tsakaki{at}pu-toyama.ac.jp

Abstract

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 1{alpha},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 1{alpha},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.


Key words: Vitamin D, Structure-activity relationships and modeling, Cytochrome P450, Enzymology


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