Structure and stereochemistry of products of hydroxylation of human steroid hormones by a housefly cytochrome P450 (CYP6A1).
The structure and stereochemistry of nine steroid metabolites isolated in quantities ranging from 0.15 to 1.8 mg were determined using a variety of NMR techniques, including heteronuclear multiple bond correlation (HMBC) using broadband adiabatic 13C pulses and phase-sensitive data presentation. Testosterone, androstenedione and progesterone were oxidized with housefly cytochrome P450 6A1 enzyme reconstituted in vitro with housefly NADPH cytochrome P450 reductase and cytochrome b5. NMR analysis in CD3OD using a modified HMBC sequence as well as 2D heteronuclear single quantum correlation (HSQC), COSY and nuclear Overhauser and exchange spectroscopy (NOESY), combined with a detailed analysis of J couplings showed that hydroxylation occurs exclusively on the beta-face of the steroids, at positions 2, 12, and 15.
Jacobsen
Neil E
NE
Department of Chemistry, University of Arizona, Tucson, AZ 85721, USA. neil@email.arizona.edu
Kövér
Katalin E
KE
Murataliev
Marat B
MB
Feyereisen
René
R
Walker
F Ann
FA
D000818:Animals; D002247:Carbon Isotopes / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis / Q000378:metabolism*; D006728:Hormones / Q000032:analysis / Q000378:metabolism*; D006793:Houseflies / Q000201:enzymology; D006801:Humans; D006900:Hydroxylation; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D015394:Molecular Structure; D013256:Steroids / Q000032:analysis / Q000378:metabolism*; D013997:Time Factors
D002247:Carbon Isotopes; D006728:Hormones; D013256:Steroids; D003577:Cytochrome P-450 Enzyme System