STEROID HYDROXYLATION BY HUMAN FETAL CYP3A7 AND HUMAN NADPH-CYTOCHROME P450 REDUCTASE COEXPRESSED IN INSECT CELLS USING BACULOVIRUS

Human fetal CYP3A7 and human NADPH-cytochrome P450 reductase were coex pressed in insect cells, TN-5, infected with a recombinant baculovirus carrying both cDNAs. The expression of reductase in TN-5 cells was sh own to be sufficient for the CYP3A7 dependent 16 alpha-hydroxylation o f dehydroepiandrosterone. However, the extra addition of cytochrome b5 and phospholipid was necessary to obtain a maximal activity of CYP3A7 catalyzing the reaction. CYP3A7 expressed in TN-5 eels was capable of metabolizing testosterone, cortisol and dehydroepiandrosterone 3-sulf ate as well as dehydroepiandrosterone. The apparent Vmax for 6 beta-hy droxylations of testosterone was similar to that obtained for 6 beta-h ydroxylation of cortisol (2.9 versus 2.5 nmol/nmolP450/min). In contra st, the apparent Vmax for 16 alpha-hydroxylation of dehydroepiandroste rone and its 3-sulfate were 20 and 2 times greater than those observed for steroid 6 beta-hydroxylations, respectively (67.5 and 5.8 versus 2.5-2.9 nmol/nmol P450/min). On the other hand, the apparent Km for 6 beta-hydroxylations of testosterone and cortisol were greater than tho se for 16 alpha-hydroxyiations (120 and 860 versus 46-58 mu M). Thus, CYP3A7 was active for steroid 6 beta-hydroxylations and 16 alpha-hydro xylations, but there were greater differences in Vm.Vmax/Km ratios bet ween these reactions.