Abstract
Cytochrome P450scc catalyses the cleavage of the side chain of both cholesterol and the vitamin D3 precursor, 7-dehydrocholesterol. The aim of this study was to test the ability of human cytochrome P450scc to metabolize ergosterol, the vitamin D2 precursor, and define the structure of the major products. P450scc incorporated into the bilayer of phospholipid vesicles converted ergosterol to two major and four minor products with a kcat of 53 mol/min/mol P450scc and a Km of 0.18 mol ergosterol/mol phospholipid, similar to the values observed for cholesterol metabolism. The reaction of ergosterol with P450scc was scaled up to make enough of the two major products for structural analysis. From mass spectrometry, NMR and comparison of the NMR data to that for similar molecules we determined the structures of the two major products as 20-hydroxy-22,23-epoxy-22,23-dihydroergosterol and 22-keto-23-hydroxy-22,23-dihydroergosterol. Molecular modeling and NOESY spectra analysis helped to establish the configurations at C-20, 22, and 23 and determine the final structures of major products as 22R,23S-epoxyergosta-5,7-diene-3β,20α-diol and 3β,23S-dihydroxyergosta-5,7-dien-22-one. It is likely that the formation of the second product is through a 22,23- epoxy (oxirane) intermediate followed by C22-hydroxylation with the formation of strained 22-hydroxy-22,23- epoxide (oxiranol) which is immediately transformed to the more stable α-hydroxyketone. Molecular modeling of ergosterol into the P450scc crystal structure positioned the ergosterol side chain consistent with formation of the above products. Thus we have shown that P450scc efficiently catalyses epoxide formation with ergosterol giving rise to novel epoxy-, hydroxy- and keto-derivatives, without causing cleavage of the side chain.
- cholesterol
- drug disposition
- enzyme kinetics
- human CYP enzymes
- liposomes
- steroids
- structure elucidation
- vitamin D
- Received August 26, 2011.
- Accepted November 21, 2011.
- The American Society for Pharmacology and Experimental Therapeutics