Generation of a homology model for the human cytochrome P450, CYP24A1, and the testing of putative substrate binding residues by site-directed mutagenesis and enzyme activity studies.
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 1alpha,25-dihydroxyvitamin D3. 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.
Masuda
Sonoko
S
Department of Biochemistry, Queen's University, Kingston, ON, Canada K7L 3N6.
Prosser
David E
DE
Guo
Yu-Ding
YD
Kaufmann
Martin
M
Jones
Glenville
G
D019943:Amino Acid Substitution; D000818:Animals; D002117:Calcitriol / Q000737:chemistry* / Q000378:metabolism*; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary; D013250:Steroid Hydroxylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity / Q000235:genetics; D065668:Vitamin D3 24-Hydroxylase
D013250:Steroid Hydroxylases; C585549:CYP24A1 protein, human; D065668:Vitamin D3 24-Hydroxylase; D002117:Calcitriol