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

Abstract

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 β-3 sheet, and the β-5 sheet. Site-directed mutagenesis and expression in COS-1 cells confirmed that these positions affect enzymatic activity, in some cases shifting metabolism of 1α-hydroxyvitamin D3 to favor 25- or 27-hydroxylation. The results suggest that conserved hydrophobic residues in the β-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 β-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.