Role of Gln 85 of human CYP27A1 in 25-hydroxyvitamin D(3)-binding and protein folding.
CYP27A1 catalyzes vitamin D(3) 25-hydroxylation and further hydroxylation at C-1alpha, C-24 or C-26(27). Molecular modeling of human CYP27A1 and docking with 25-hydroxyvitamin D(3) predicted that Gln 85 might be important for 1alpha-hydroxylation activity of CYP27A1 by forming a hydrogen bond with the 25-OH group of 25-hydroxyvitamin D(3). Expectedly, the mutant Q85H expressed in Escherichia coli showed no detectable 1alpha-hydroxylation activity toward 25-hydroxyvitamin D(3). In addition, Q85H prefers 24-hydroxylation toward 25-hydroxyvitamin D(3) whereas the wild-type prefers 26(27)-hydroxylation. A molecular modeling study also suggests that Gln 85 of CYP27A1 simultaneously interacts with Asn 107 and the hydroxyl group of the substrate. The fact that Q85L did not contain a heme molecule suggests that the hydrogen bond between Gln 85 and Asn 107 is important for protein folding of CYP27A1. Based on these results, it is possible that Gln 85 plays essential roles in both substrate-binding and protein folding.
Sawada
Natsumi
N
Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
Yamamoto
Keiko
K
Yamada
Sachiko
S
Ikushiro
Shinichi
S
Kamakura
Masaki
M
Ohta
Miho
M
Inouye
Kuniyo
K
Sakaki
Toshiyuki
T
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D002112:Calcifediol / Q000378:metabolism*; D053493:Cholestanetriol 26-Monooxygenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005973:Glutamine; D006801:Humans; D006900:Hydroxylation; D008958:Models, Molecular; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D014162:Transfection
D017931:DNA Primers; D011994:Recombinant Proteins; D005973:Glutamine; C506831:CYP27A1 protein, human; D053493:Cholestanetriol 26-Monooxygenase; D002112:Calcifediol