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

Ram P. Gupta, You Ai He, Kennerly S. Patrick, James R. Halpert, Norman H. Bell
2005 Journal of Clinical Endocrinology and Metabolism  
Running title: CYP3A4 vitamin D hydroxylase structure function Key words: vitamin D 2 , 1α-hydroxyvitamin D 2 , 1α-hydroxyvitamin D 3 , 24-hydroxylase, 25hydroxylase, site-directed mutagenesis. These studies were supported by grants DK56603-01A1 and GM 54995 from the National Institutes of Health. Abstract 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
more » ... osomal 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 1α-hydroxyvitamin D 2 [1αOHD 2 ] as substrate. The ratio of rates of 24 hydroxylation of 1α-hydroxyvitamin D 3 [1αOHD 3 ], 1αOHD 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 1αOHD 2 by CYP3A4 correlated significantly with 6β-hydroxylation of testosterone, a marker of CYP3A4 activity. 24-Hydroxylase activity in recombinant CYP3A4 and pooled human liver microsomes showed dosedependent inhibition by ketoconazole, troleandomycin, -naphthoflavone, and isoniazid, known inhibitors of CYP3A4. Rates of 24-and 25-hydroxylation of 1αOHD 2 and 1αOHD 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 , 1αOHD 2 , and 1αOHD 3 . µl of microsomes from baculovirus-infected insect cells and 100 µl of 0.5 M Na 2 HPO4 (pH 7.4), 50 µl of 60 mM EDTA, 50 µl of NADPH regenerating solution A (BD Gentest), 10 µl of regenerating solution B (BD Gentest), 2 µl of 5 mM dianilinoethane and 2 µl of 1αOHD 2 , 1αOHD 3 , or vitamin D 2 . The final concentration of substrate was 10 µM. Unless otherwise indicated, the reaction was performed at 37 o C for 1.5 h, and terminated with the addition of 1 ml of acetonitrile. Protein was measured by the bicinchoninic acid method with a kit (Pierce, Rockford, IL) (12). Construction, expression, and purification of CYP3A4 and mutants Plasmid pE3A4His was used to express CYP3A4. Details of construction of CYP3A4 mutants (13-15) and of naturally occurring CYP3A4 protein variants (16) were as previously described. All constructs were transformed into E. coli strain DH5a or Topp3 (Stratagene, La Jolla, CA). Transformed cells were grown overnight on a Luria Bertani (LB) plate containing 50 µg/ml ampicillin at 37 o C. A single colony was inoculated in 20 ml of LB broth with 50 µg/ml ampicillin. After overnight growth at 37 o C with vigorous shaking, 15 ml of culture was transferred into a 1-L flask containing 250 ml of tryptone broth with 100 µg/ml ampicillin. Cultures were grown at 37 o C with shaking at 250 rpm. When cultures had an absorbance of 1.2 at 550 nm, 1 mM isopropylthio-β-D-galactoside and 80 mg/L of ALA were added. Cells were harvested after an additional 72-h incubation at 30 o C with shaking at 190 rpm. CHAPS-solubilized membrane preparations were made as described previously (17). HIS-tagged proteins were purified by column chromatography with TALON metal affinity resin (CLONTECH, Palo Alto, CA) (16). P450 content was determined by carbon monoxide difference spectra in the presence of 1% Triton X-100 added to the protein sample before dilution with microsome-solubilization buffer containing 100 mM potassium phosphate (pH 7.3), 20% glycerol, 0.5% sodium chrome P450 mRNA molecules using competitive reverse transcriptase-PCR. DNA Cell Biol 17:231-238 31. Edwards RJ, Adams DA, Watts PS, Davies DS, Boobis AR.1998 Development of a comprehensive panel of antibodies against the major xenobiotic metabolizing forms of cytochrome P450 in humans. Biochem Pharmacol 56:177-187 21 32. 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doi:10.1210/jc.2004-0966 pmid:15546903 fatcat:fd34qh72sngj3pmnmiql4xbrjm