CYP3A4 is a vitamin D-24- and 25-hydroxylase: analysis of structure function by site-directed mutagenesis.
Studies were performed to identify the microsomal enzyme that 24-hydroxylates vitamin D, whether 25-hydroxylation occurs, and structure function of the enzyme. Sixteen hepatic recombinant microsomal cytochrome P450 enzymes expressed in baculovirus-infected insect cells were screened for 24-hydroxylase activity. CYP3A4, a vitamin D-25-hydroxylase, and CYP1A1 had the highest 24-hydroxylase activity with 1 alpha-hydroxyvitamin D(2) (1 alpha OHD(2)) as substrate. The ratio of rates of 24-hydroxylation of 1 alpha-hydroxyvitamin D(3) (1 alpha OHD(3)), 1 alpha OHD(2), and vitamin D(2) by CYP3A4 was 3.6/2.8/1.0. Structures of 24-hydroxyvitamin D(2), 1,24(S)-dihydroxyvitamin D(2), and 1,24-dihydroxyvitamin D(3) were confirmed by HPLC and gas chromatography retention time and mass spectroscopy. In characterized human liver microsomes, 24-hydroxylation of 1 alpha OHD(2) by CYP3A4 correlated significantly with 6 beta-hydroxylation of testosterone, a marker of CYP3A4 activity. 24-Hydroxylase activity in recombinant CYP3A4 and pooled human liver microsomes showed dose-dependent inhibition by ketoconazole, troleandomycin, alpha-naphthoflavone, and isoniazid, known inhibitors of CYP3A4. Rates of 24- and 25-hydroxylation of 1 alpha OHD(2) and 1 alpha OHD(3) were determined in recombinant wild-type CYP3A4 and site-directed mutants and naturally occurring variants expressed in Escherichia coli. Substitution of residues showed the most prominent alterations of function at residues 119, 120, 301, 305, and 479. Thus, CYP3A4 is both a 24- and 25-hydroxylase for vitamin D(2), 1 alpha OHD(2), and 1 alpha OHD(3).
Gupta
Ram P
RP
Department of Medicine, Medical University of South Carolina, P.O. Box 250775, Charleston, South Carolina 29425, USA.
He
You Ai
YA
Patrick
Kennerly S
KS
Halpert
James R
JR
Bell
Norman H
NH
D000818:Animals; D016367:Baculoviridae; D053493:Cholestanetriol 26-Monooxygenase; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D006801:Humans; D007313:Insecta; D007538:Isoniazid / Q000494:pharmacology; D007654:Ketoconazole / Q000494:pharmacology; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000378:metabolism; D013250:Steroid Hydroxylases / Q000235:genetics*; D014162:Transfection; D014217:Troleandomycin / Q000494:pharmacology; D014807:Vitamin D / Q000378:metabolism*; D065668:Vitamin D3 24-Hydroxylase
D011994:Recombinant Proteins; D014807:Vitamin D; D003577:Cytochrome P-450 Enzyme System; D014217:Troleandomycin; D013250:Steroid Hydroxylases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; C506831:CYP27A1 protein, human; D053493:Cholestanetriol 26-Monooxygenase; D065668:Vitamin D3 24-Hydroxylase; D007654:Ketoconazole; D007538:Isoniazid