Functional studies of the human 25-hydroxyvitamin D3-24-hydroxylase, CYP24A1

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Kaufmann, Martin
vitamin D , metabolism , cytochrome P450 , CYP24A1 , Vitamin D analog
This thesis focuses on the cytochrome P450 CYP24A1, which catabolizes 1α,25-dihydroxyvitamin D3 (1α,25-(OH)2D3) via two pathways commencing with C24-, or C23-hydroxylation and culminating in calcitroic acid or 1α,25-(OH)2D3-26,23-lactone respectively. In my thesis, I set out to identify the molecular determinants of the unique multicatalytic activity of human (h) CYP24A1, using a combination of techniques including homology modeling, site-directed mutagenesis and enzyme activity assays. In total, 42 hCYP24A1 mutants were stably transfected into V79-4 cells, and the metabolism of 1α,25-(OH)2D3 and 9 analogs or prodrugs were studied using HPLC and LC-MS-based techniques. Overall, we observed that mutations at sites in contact with the side-chain of 1α,25-(OH)2D3 above the heme in the homology model, affected the regioselectivity of the protein, and mutations at more distal sites along the substrate cavity affected the access of the substrate or intermediates. Of these mutations, A326G and V391L were of particular interest. Ala326, in the centre of the I-helix in contact with the side chain of 1α,25-(OH)2D3, is a major determinant of the regioselectivity difference between opossum (C23-hydroxylating) and human (C24-hydroxylating) enzymes. When Ala326 was mutated to Gly, as it is in opossum, hCYP24A1 was converted from a C24-hydroxylase, to a C23-hydroxylase, forming 1α,25-(OH)2D3-26,23-lactone. We propose that A326G permits the side chain to enter the substrate binding site more deeply, thus positioning C23 as opposed to C24 above the heme for hydroxylation. Another mutation, V391L in the beta-3a sheet, drastically changed the substrate specificity of CYP24A1 towards the prodrug 1α-hydroxyvitamin D3 (1α-OH-D3), which is not metabolized by the wild-type enzyme. V391L converted hCYP24A1 into a 1α-OH-D3-25-hydroxylase, forming 1α,25-(OH)2D3 which was subsequently catabolized via C24-hydroxylation by the same enzyme. The downstream catabolism of 1α,25-(OH)2D3 formed from 1α-OH-D3 was engineered to proceed via C23-hydroxylation, by introducing a V391L/A326G double mutant. We propose that altered steric contact at C21 is responsible for enabling C25-hydroxylation by V391L. Taken together, we have successfully developed an approach that has identified key structural and functional insights into the mechanism of action of hCYP24A1 that can be potentially applied to study other cytochrome P450 enzymes.
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