Journal
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Volume 460, Issue 2, Pages 177-191Publisher
ELSEVIER SCIENCE INC
DOI: 10.1016/j.abb.2006.11.030
Keywords
vitamin D; calcitriol; cytochrome P450; CYP24A1; homology modeling; 1 alpha,25(OH)(2)D-3; mutagenesis; vitamin D dependent-gene expression; vitamin D analogs; CYP24A1 inhibitors
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A systematic analysis of conserved H-bonding patterns and tertiary structural motifs from 13 crystal structures was used to create a homology model for the human multicatalytic cytochrome P450, CYP24A1, involved in catabolism of 1 alpha,25-dihydroxyvitamin D-3. The substrate was docked in the active site and used to identify potential substrate contact residues in the B' helix, B'/C loop, F-helix and the beta-5 hairpin. Seven CYP24A1 mutants were created and studied by mammalian cell transfection and CYP24A1 activity assay. Mutants showed reduced metabolic rates and altered metabolite patterns compared to wild-type. We conclude that: Ile-131 positions substrate via A-ring and cis-triene contacts; Trp-134 and Gly-499 are determinants of substrate access; Leu-148 contacts the substrate side-chain; Met-246 is important in mediating regioselectivity. Our findings validate the new model of CYP24A1, which can now be used to predict structural modifications for rational vitamin D drug design. (c) 2006 Elsevier Inc. All rights reserved.
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