title: Metabolism of 1alpha-hydroxyvitamin D3 by cytochrome P450scc to biologically active 1alpha,20-dihydroxyvitamin D3.
abstract: Cytochrome P450scc (CYP11A1) metabolizes vitamin D3 to 20-hydroxyvitamin D3 as the major product, with subsequent production of dihydroxy and trihydroxy derivatives. The aim of this study was to determine whether cytochrome P450scc could metabolize 1alpha-hydroxyvitamin D3 and whether products were biologically active. The major product of 1alpha-hydroxyvitamin D3 metabolism by P450scc was identified by mass spectrometry and NMR as 1alpha,20-dihydroxyvitamin D3. Mass spectrometry of minor metabolites revealed the production of another dihydroxyvitamin D3 derivative, two trihydroxy-metabolites made via 1alpha,20-dihydroxyvitamin D3 and a tetrahydroxyvitamin D3 derivative. The Km for 1alpha-hydroxyvitamin D3 determined for P450scc incorporated into phospholipid vesicles was 1.4 mol substrate/mol phospholipid, half that observed for vitamin D3. The kcat was 3.0 mol/min/mol P450scc, 6-fold lower than that for vitamin D3. 1alpha,20-Dihydroxyvitamin D3 inhibited DNA synthesis by human epidermal HaCaT keratinocytes propagated in culture, in a time- and dose-dependent fashion, with a potency similar to that of 1alpha,25-dihydroxyvitamin D3. 1alpha,20-Dihydroxyvitamin D3 (10 microM) enhanced CYP24 mRNA levels in HaCaT keratinocytes but the potency was much lower than that reported for 1alpha,25-dihydroxyvitamin D3. We conclude that the presence of the 1-hydroxyl group in vitamin D3 does not alter the major site of hydroxylation by P450scc which, as for vitamin D3, is at C20. The major product, 1alpha,20-dihydroxyvitamin D3, displays biological activity on keratinocytes and therefore might be useful pharmacologically.
authors.affiliation: School of Biomedical, Biomolecular and Chemical Sciences, M310, The University of Western Australia, Crawley, WA 6009, Australia. rtuckey@cyllene.uwa.edu.au
authors.forename: Robert C
authors.initials: RC
authors.lastname: Tuckey
authors.forename: Zorica
authors.initials: Z
authors.lastname: Janjetovic
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.forename: Minh N
authors.initials: MN
authors.lastname: Nguyen
authors.forename: Michal A
authors.initials: MA
authors.lastname: Zmijewski
authors.forename: Jordan
authors.initials: J
authors.lastname: Zjawiony
authors.forename: Andrzej
authors.initials: A
authors.lastname: Slominski
mesh_terms: D000818:Animals; D002417:Cattle; D049109:Cell Proliferation / Q000187:drug effects; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D006887:Hydroxycholecalciferols / Q000096:biosynthesis* / Q000378:metabolism* / Q000494:pharmacology; D015603:Keratinocytes / Q000166:cytology; D007700:Kinetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D013250:Steroid Hydroxylases / Q000096:biosynthesis; D065668:Vitamin D3 24-Hydroxylase
chemical_list: C539020:1alpha,20-dihydroxyvitamin D3; D006887:Hydroxycholecalciferols; D013250:Steroid Hydroxylases; D065668:Vitamin D3 24-Hydroxylase; D002786:Cholesterol Side-Chain Cleavage Enzyme; C008088:alfacalcidol