Edited by Hector F. DeLuca, University of Wisconsin, Madison, WI, and approved June 18, 2007 (received for review March 8, 2007)

Studies of 25-hydroxyvitamin D₃-24-hydroxylase (CYP24A1) have demonstrated that it is a bifunctional enzyme capable of the 24-hydroxylation of 1,25-(OH)₂D₃, leading to the excretory form, calcitroic acid, and 23-hydroxylation, culminating in 1,25-(OH)₂D₃-26,23-lactone. The degree to which CYP24A1 performs either 23- or 24-hydroxylation is species-dependent. In this paper, we show that the human enzyme that predominantly 24-hydroxylates its substrate differs from the opossum enzyme that 23-hydroxylates it at only a limited number of amino acid residues. Mutagenesis of the human form at a single substrate-binding residue (A326G) dramatically changes the regioselectivity of the enzyme from a 24-hydroxylase to a 23-hydroxylase, whereas other modifications have no effect. Ala-326 is located in the I-helix, close to the terminus of the docked 25-hydroxylated side chain in a CYP24A1 homology model, a result that we interpret indicates that substitution of a glycine at 326 provides extra space for the side chain of the substrate to move deeper into the pocket and place it in a optimal stereochemical position for 23-hydroxylation. We discuss the physiological ramifications of these results for species possessing the A326G substitution, as well as implications for optimal vitamin D analog design.