Abstract
Cytochrome P450scc (P450scc) catalyzes 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 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−1 · mol P450scc−1 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 nuclear Overhauser effect (or enhancement) spectroscopy spectra analysis helped to establish the configurations at C20, C22, and C23 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 catalyzes epoxide formation with ergosterol giving rise to novel epoxy, hydroxy, and keto derivatives, without causing cleavage of the side chain.
Footnotes
This work was supported by the National Institutes of Health National Institute of Arthritis and Musculoskeletal and Skin Diseases [Grant R01-AR052190] (to A.T.S.); the University of Western Australia; and the College of Pharmacy at the University of Tennessee Health Science Center.
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material.
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ABBREVIATIONS:
- P450scc
- cytochrome P450scc
- COSY
- correlation spectroscopy
- TOCSY
- total correlation spectroscopy
- NOESY
- nuclear Overhauser effect spectroscopy
- HSQC
- heteronuclear single quantum correlation spectroscopy
- HMBC
- heteronuclear multiple-bond correlation spectroscopy
- TLC
- thin-layer chromatography
- HPLC
- high-performance liquid chromatography
- NOE
- nuclear Overhauser effect.
- Received August 25, 2011.
- Accepted November 21, 2011.
- Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
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