title: Bioengineering anabolic vitamin D-25-hydroxylase activity into the human vitamin D catabolic enzyme, cytochrome P450 CYP24A1, by a V391L mutation.
abstract: CYP24A1 is a mitochondrial cytochrome P450 (CYP) that catabolizes 1α,25-dihydroxyvitamin D(3) (1α,25-(OH)(2)D(3)) to different products: calcitroic acid or 1α,25-(OH)(2)D(3)-26,23-lactone via multistep pathways commencing with C24 and C23 hydroxylation, respectively. Despite the ability of CYP24A1 to catabolize a wide range of 25-hydroxylated analogs including 25-hydroxyvitamin D(3), the enzyme is unable to metabolize the synthetic prodrug, 1α-hydroxyvitamin D(3) (1α-OH-D(3)), presumably because it lacks a C25-hydroxyl. In the current study we show that a single V391L amino acid substitution in the β3a-strand of human CYP24A1 converts this enzyme from a catabolic 1α,25-(OH)(2)D(3)-24-hydroxylase into an anabolic 1α-OH-D(3)-25-hydroxylase, thereby forming the hormone, 1α,25-(OH)(2)D(3). Furthermore, because the mutant enzyme retains its basal ability to catabolize 1α,25-(OH)(2)D(3) via C24 hydroxylation, it can also make calcitroic acid. Previous work has shown that an A326G mutation is responsible for the regioselectivity differences observed between human (primarily C24-hydroxylating) and opossum (C23-hydroxylating) CYP24A1. When the V391L and A326G mutations were combined (V391L/A326G), the mutant enzyme continued to form 1α,25-(OH)(2)D(3) from 1α-OH-D(3), but this initial product was diverted via the C23 hydroxylation pathway into the 26,23-lactone. The relative position of Val-391 in the β3a-strand of a homology model and the crystal structure of rat CYP24A1 is consistent with hydrophobic contact of Val-391 and the substrate side chain near C21. We interpret that the substrate specificity of V391L-modified human CYP24A1 toward 1α-OH-D(3) is enabled by an altered contact with the substrate side chain that optimally positions C25 of the 1α-OH-D(3) above the heme for hydroxylation.
authors.affiliation: Department of Biochemistry, Queen's University, Kingston, Ontario, Canada K7L 3N6.
authors.forename: Martin
authors.initials: M
authors.lastname: Kaufmann
authors.affiliation: Department of Biochemistry, Queen's University, Kingston, Ontario, Canada K7L 3N6.
authors.forename: David E
authors.initials: DE
authors.lastname: Prosser
authors.affiliation: Department of Biochemistry, Queen's University, Kingston, Ontario, Canada K7L 3N6; Department of Medicine, Queen's University, Kingston, Ontario, Canada K7L 3N6. Electronic address: gj1@queensu.ca.
authors.forename: Glenville
authors.initials: G
authors.lastname: Jones
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002112:Calcifediol / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D053493:Cholestanetriol 26-Monooxygenase; D006224:Cricetinae; D006801:Humans; D006900:Hydroxylation / Q000235:genetics; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D051381:Rats; D013250:Steroid Hydroxylases / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity / Q000235:genetics; D065668:Vitamin D3 24-Hydroxylase
chemical_list: D024101:Mitochondrial Proteins; D013250:Steroid Hydroxylases; D053493:Cholestanetriol 26-Monooxygenase; C585549:CYP24A1 protein, human; C585551:Cyp24a1 protein, rat; D065668:Vitamin D3 24-Hydroxylase; D002112:Calcifediol