title: Single A326G mutation converts human CYP24A1 from 25-OH-D3-24-hydroxylase into -23-hydroxylase, generating 1alpha,25-(OH)2D3-26,23-lactone.
abstract: Studies of 25-hydroxyvitamin D(3)-24-hydroxylase (CYP24A1) have demonstrated that it is a bifunctional enzyme capable of the 24-hydroxylation of 1alpha,25-(OH)(2)D(3), leading to the excretory form, calcitroic acid, and 23-hydroxylation, culminating in 1alpha,25-(OH)(2)D(3)-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.
authors.affiliation: Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
authors.forename: David E
authors.initials: DE
authors.lastname: Prosser
authors.forename: Martin
authors.initials: M
authors.lastname: Kaufmann
authors.forename: Brendan
authors.initials: B
authors.lastname: O'Leary
authors.forename: Valarie
authors.initials: V
authors.lastname: Byford
authors.forename: Glenville
authors.initials: G
authors.lastname: Jones
mesh_terms: D000409:Alanine / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002117:Calcitriol / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D002384:Catalysis; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D017124:Conserved Sequence; D006224:Cricetinae; D003412:Cricetulus; D005786:Gene Expression Regulation; D005998:Glycine / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009154:Mutation / Q000235:genetics; D016415:Sequence Alignment; D013250:Steroid Hydroxylases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D065668:Vitamin D3 24-Hydroxylase
chemical_list: C026947:1,25-dihydroxyvitamin D3-26,23-lactone; D013250:Steroid Hydroxylases; C585549:CYP24A1 protein, human; D065668:Vitamin D3 24-Hydroxylase; D002117:Calcitriol; D000409:Alanine; D005998:Glycine