Single A326G mutation converts human CYP24A1 from 25-OH-D3-24-hydroxylase into -23-hydroxylase, generating 1α,25-(OH)2D3-26,23-lactone
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Edited by Hector F. DeLuca, University of Wisconsin, Madison, WI, and approved June 18, 2007 (received for review March 8, 2007)
Abstract
Studies of 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1) have demonstrated that it is a bifunctional enzyme capable of the 24-hydroxylation of 1α,25-(OH)2D3, leading to the excretory form, calcitroic acid, and 23-hydroxylation, culminating in 1α,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.
Footnotes
- *To whom correspondence should be addressed at: Department of Biochemistry, Queen's University, Botterell Hall, Room 650, Kingston, ON, Canada K7L 3N6. E-mail: gj1{at}queensu.ca
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Author contributions: D.E.P. and M.K. contributed equally to this work; D.E.P., M.K., and G.J. designed research; D.E.P., M.K., B.O., and V.B. performed research; D.E.P., M.K., and G.J. contributed new reagents/analytic tools; D.E.P., M.K., B.O., V.B., and G.J. analyzed data; and D.E.P., M.K., and G.J. wrote the paper.
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The authors declare no conflict of interest.
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This article is a PNAS Direct Submission.
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This article contains supporting information online at www.pnas.org/cgi/content/full/0702093104/DC1.
- Abbreviations:
- 1α,25-(OH)2D3,
- 1α,25-dihydroxyvitamin D3;
- 25-OH-D3,
- 25-hydroxyvitamin-D3;
- CYP,
- cytochrome P450;
- VDR,
- 1α,25-dihydroxyvitamin D3 receptor;
- OK,
- opossum kidney;
- LC,
- liquid chromatography;
- sp.,
- specific;
- GAA,
- glacial acetic acid.
- © 2007 by The National Academy of Sciences of the USA
