Pregnenolone-7β-hydroxylating activities of yeast-expressed mouse cytochrome P450-1A1 and mouse-tissue microsomes

Abstract

In many tissues from different species, pregnenolone and dehydroepiandrosterone (DHEA) are hydroxylated mainly at the 7α position by a cytochrome P450 (P450)-containing microsomal enzyme complex. In addition, 7-hydroxysteroids have been shown to activate immune processes in mice. The reported production of 7β-hydroxypregnenolone and 7β-hydroxy-DHEA was not supported by formal identification, and the P450 responsible for 7α-hydroxylation and 7β-hydroxylation of pregnenolone and DHEA have not been identified. Based on results of analyses by crystallization to constant specific activity and gas chromatography/mass spectrometry, we report that mouse-liver and mouse-brain microsomes carried out 7β-hydroxylation of pregnenolone and DHEA, and that yeast-expressed mouse cytochrome P450-1A1 (P450 1A1) transformed pregnenolone into 7β-hydroxypregnenolone (K(m) 25.1 ± 0.4 μM, turnover number = 979 ± 30 pmol · min-1 · nmol-1 mouse P450 1A1). Neither 7-hydroxy derivatives of DHEA nor 7α-hydroxypregnenolone was produced by P450 1A1. The presence of P450 1A1 in liver and brain microsomes was shown by Western blot analysis, and induction of mouse P450 1A1 by β-naphthoflavone resulted in increased 7β-hydroxylation of pregnenolone in liver microsomes. Studies of the brain-microsome 7β-hydroxylating enzyme with pregnenolone or DHEA gave K(m) of 5.0 μM and 4.9 μM, respectively, and V(max) of 4.5 pmol · min-1 · mg-1 and 6.1 pmol · min-1 · mg-1, respectively, and showed the absence of cross-inhibitions between the two steroids. These findings indicate that, in addition to unidentified P450, P450 1A1 is involved in 7β-hydroxylation of pregnenolone and may contribute in part to the production of the 7-hydroxylated steroids necessary for activation of immune defenses.