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Structural Motif-Based Homology Modeling of CYP27A1 and Site-Directed Mutational Analyses Affecting Vitamin D Hydroxylation

Department of Biochemistry, Queen's University, Kingston, Ontario, Canada

Correspondence: Address reprint requests to Dr. Glenville Jones, Queen's University, Dept. of Biochemistry, Botterell Hall, Room 277, Kingston, Ontario, Canada K7L 3N6. Tel: 613-533-2498; Fax: 613-533-2987; E-mail: gj1{at}post.queensu.ca.

Human CYP27A1 is a mitochondrial cytochrome P450, which is principally found in the liver and plays important roles in the biological activation of vitamin D₃ 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 D₃ structures into the active site of this model identified potential substrate contact residues in the F-helix, the ß-3 sheet, and the ß-5 sheet. Site-directed mutagenesis and expression in COS-1 cells confirmed that these positions affect enzymatic activity, in some cases shifting metabolism of 1-hydroxyvitamin D₃ to favor 25- or 27-hydroxylation. The results suggest that conserved hydrophobic residues in the ß-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 ß-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.