Metabolism of vitamin D3 by cytochromes P450

The vitamin D-3 25-hydroxylase (CYP27A1), 25-hydroxyvitamin D-3 1alpha-hydroxylase (CYP27B1) and 1alpha, 25-dihydroxyvitamin D-3 24-hydroxylase (CYP24A1) are members of the cytochrome P450 superfamily, and key enzymes of vitamin D-3 metabolism. Using the heterologous expression in E. coli, enzymatic properties of the P450s were recently investigated in detail. Upon analyses of the metabolites of vitamin D-3 by the reconstituted system, CYP27A1 surprisingly produced at least seven forms of minor metabolites including 1alpha, 25( OH)(2)D-3 in addition to the major metabolite 25( OH)D-3. These results indicated that human CYP27A1 catalyzes multiple reactions involved in the vitamin D-3 metabolism. In contrast, CYP27B1 only catalyzes the hydroxylation at C-1alpha position of 25(OH) D-3 and 24R, 25(OH)(2)D-3. Enzymatic studies on substrate specificity of CYP27B1 suggest that the 1alpha-hydroxylase activity of CYP27B1 requires the presence of 25-hydroxyl group of vitamin D-3 and is enhanced by 24-hydroxyl group while the presence of 23-hydroxyl group greatly reduced the activity. Eight types of missense mutations in the CYP27B1 gene found in vitamin D-dependent rickets type I (VDDR-I) patients completely abolished the 1alpha-hydroxylase activity. A three-dimensional model of CYP27B1 structure simulated on the basis of the crystal structure of rabbit CYP2C5 support s the experimental data from mutagenesis study of CYP27B1 that the mutated amino acid residues may be involved in protein folding, heme-propionate binding or activation of molecular oxygen. CYP24A1 expressed in E. coli showed a remarkable metabolic processes of 25(OH) D-3 and 1alpha, 25( OH)(2)D-3. Rat CYP24A1 catalyzed six sequential monooxygenation reactions that convert 1alpha, 25(OH)(2)D-3 into calcitroic acid, a known final metabolite of C-24 oxidation pathway. In addition to the C-24 oxidation pathway, human CYP24A1 catalyzed also C-23 oxidation pathway to produce 1alpha, 25(OH)(2)D-3- 26,23-lactone. Surprisingly, more than 70 % of the vitamin D metabolites observed in a living body were found to be the products formed by the activities of CYP27A1, CYP27B1 and CYP24A1. The species-based difference was also observed in the metabolism of vitamin D analogs by CYP24A1, suggesting that the recombinant system for human CYP24A1 may be of great use for the prediction of the metabolism of vitamin D analogs in humans.