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.
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 1alpha,25-dihydroxyvitamin D3 [1alpha,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 1alpha,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.
Hamamoto
Hiromi
H
Biotechnology Research Center, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.
Kusudo
Tatsuya
T
Urushino
Naoko
N
Masuno
Hiroyuki
H
Yamamoto
Keiko
K
Yamada
Sachiko
S
Kamakura
Masaki
M
Ohta
Miho
M
Inouye
Kuniyo
K
Sakaki
Toshiyuki
T
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002117:Calcitriol / Q000378:metabolism; D020134:Catalytic Domain / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007532:Isoleucine / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013250:Steroid Hydroxylases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013912:Threonine / Q000235:genetics / Q000378:metabolism; D065668:Vitamin D3 24-Hydroxylase
D000596:Amino Acids; D050505:Mutant Proteins; D007532:Isoleucine; D013912:Threonine; D013250:Steroid Hydroxylases; C585549:CYP24A1 protein, human; C585551:Cyp24a1 protein, rat; D065668:Vitamin D3 24-Hydroxylase; D002117:Calcitriol