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Biophys. J. BioFAST: First Published February 24, 2006. doi:10.1529/biophysj.105.069369
© 2006 by the Biophysical Society.

A more recent version of this article appeared on May 15, 2006.
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BIOPHYSICAL THEORY AND MODELING

Structural Motif-based Homology Modeling of CYP27A1 and Site-directed Mutational Analyses Affecting Vitamin D Hydroxylation

David E. Prosser 1, YuDing Guo 1, Zongchao Jia 1 and Glenville Jones 1*

1 Department of Biochemistry, Queen's University at Kingston

* To whom correspondence should be addressed. E-mail: gj1{at}post.queensu.ca.

Submitted on June 23, 2005
Revised on August 1, 2005
Accepted on 19 January 2006


  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 eleven 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 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 1{alpha}-hydroxyvitamin D3 to favour 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 Phe248 in the F-helix. Mutations directed towards the beta-3a strand suggested a possible heme-binding interaction centered on Asn403 and a structural role for a substrate contact residue, Ser404.

Key Words: 25-hydroxylase, Mitochondrial P450, molecular modeling., prodrug metabolism






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Copyright © 2006 by the Biophysical Society.