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CYP3A4 Is a Vitamin D-24- and 25-Hydroxylase: Analysis of Structure Function by Site-Directed Mutagenesis

Departments of Medicine (R.P.G., N.H.B.) and Pharmaceutical Sciences (K.S.P.), Medical University of South Carolina, Charleston, South Carolina 29425; and Department of Pharmacology and Toxicology (Y.A.H., J.R.H.), University of Texas Medical Branch, Galveston, Texas 77555-1031

Address all correspondence and requests for reprints to: Norman H. Bell, M.D., Department of Medicine, Medical University of South Carolina, Strom Thurmond Research Building, 114 Doughty Street, P.O. Box 250775, Charleston, South Carolina 29425. E-mail: belln{at}musc.edu.

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-hydroxyvitamin D₂ (1OHD₂) as substrate. The ratio of rates of 24-hydroxylation of 1-hydroxyvitamin D₃ (1OHD₃), 1OHD₂, and vitamin D₂ by CYP3A4 was 3.6/2.8/1.0. Structures of 24-hydroxyvitamin D₂, 1,24(S)-dihydroxyvitamin D₂, and 1,24-dihydroxyvitamin D₃ were confirmed by HPLC and gas chromatography retention time and mass spectroscopy. In characterized human liver microsomes, 24-hydroxylation of 1OHD₂ by CYP3A4 correlated significantly with 6ß-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, -naphthoflavone, and isoniazid, known inhibitors of CYP3A4. Rates of 24- and 25-hydroxylation of 1OHD₂ and 1OHD₃ 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 D₂, 1OHD₂, and 1OHD₃.

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