Identification of the amino acid residue of CYP27B1 responsible for binding of 25-hydroxyvitamin D3 whose mutation causes vitamin D-dependent rickets type 1.
We previously reported the three-dimensional structure of human CYP27B1 (25-hydroxyvitamin D3 1alpha-hydroxylase) constructed by homology modeling. Using the three-dimensional model we studied the docking of the substrate, 25-hydroxyvitamin D3, into the substrate binding pocket of CYP27B1. In this study, we focused on the amino acid residues whose point mutations cause vitamin D-dependent rickets type 1, especially unconserved residues among mitochondrial CYPs such as Gln65 and Thr409. Recently, we successfully overexpressed mouse CYP27B1 by using a GroEL/ES co-expression system. In a mutation study of mouse CYP27B1 that included spectroscopic analysis, we concluded that in a 1alpha-hydroxylation process, Ser408 of mouse CYP27B1 corresponding to Thr409 of human CYP27B1 forms a hydrogen bond with the 25-hydroxyl group of 25-hydroxyvitamin D3. This is the first report that shows a critical amino acid residue recognizing the 25-hydroxyl group of the vitamin D3.
Institute of Biomaterials and Bioengineering & School of Biomedical Sciences, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan.
Keiko
K
Yamamoto
Eriko
E
Uchida
Naoko
N
Urushino
Toshiyuki
T
Sakaki
Norio
N
Kagawa
Natsumi
N
Sawada
Masaki
M
Kamakura
Shigeaki
S
Kato
Kuniyo
K
Inouye
Sachiko
S
Yamada
D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002112:Calcifediol / Q000378:metabolism*; D002762:Cholecalciferol / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D005973:Glutamine / Q000737:chemistry; D006801:Humans; D006860:Hydrogen Bonding; D007700:Kinetics; D051379:Mice; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D012279:Rickets / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D013379:Substrate Specificity; D013912:Threonine / Q000737:chemistry; D013997:Time Factors; D014807:Vitamin D / Q000378:metabolism*
D009841:Oligonucleotides; D011994:Recombinant Proteins; D005973:Glutamine; D014807:Vitamin D; D002762:Cholecalciferol; D013912:Threonine; D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase; D002112:Calcifediol