Structural motif-based homology modeling of CYP27A1 and site-directed mutational analyses affecting vitamin D hydroxylation.
Human CYP27A1 is a mitochondrial cytochrome P450, which is principally found in the liver and plays important roles in the biological activation of vitamin D(3) and in the biosynthesis of bile acids. We have applied a systematic analysis of hydrogen bonding patterns in 11 prokaryotic and mammalian CYP crystal structures to construct a homology-based model of CYP27A1. Docking of vitamin D(3) structures into the active site of this model identified potential substrate contact residues in the F-helix, the beta-3 sheet, and the beta-5 sheet. Site-directed mutagenesis and expression in COS-1 cells confirmed that these positions affect enzymatic activity, in some cases shifting metabolism of 1alpha-hydroxyvitamin D(3) to favor 25- or 27-hydroxylation. The results suggest that conserved hydrophobic residues in the beta-5 hairpin help define the shape of the substrate binding cavity and that this structure interacts with Phe-248 in the F-helix. Mutations directed toward the beta-3a strand suggested a possible heme-binding interaction centered on Asn-403 and a structural role for substrate contact residues Thr-402 and Ser-404.
Prosser
David E
DE
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
Guo
Yuding
Y
Jia
Zongchao
Z
Jones
Glenville
G
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D053493:Cholestanetriol 26-Monooxygenase; D003198:Computer Simulation; D004789:Enzyme Activation; D006900:Hydroxylation; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein / Q000379:methods*; D017386:Sequence Homology, Amino Acid; D013250:Steroid Hydroxylases / Q000737:chemistry*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014807:Vitamin D / Q000737:chemistry*
D014807:Vitamin D; D013250:Steroid Hydroxylases; C506831:CYP27A1 protein, human; D053493:Cholestanetriol 26-Monooxygenase