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 coexpressed in insect cells, TN-5, infected with a recombinant baculovirus carrying both cDNAs. The expression of reductase in TN-5 cells was shown to be sufficient for the CYP3A7 dependent 16 alpha-hydroxylation of 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 cells was capable of metabolizing testosterone, cortisol and dehydroepiandrosterone 3-sulfate as well as dehydroepiandrosterone. The apparent Vmax for 6 beta-hydroxylations of testosterone was similar to that obtained for 6 beta-hydroxylation of cortisol (2.9 versus 2.5 nmol/nmolP450/min). In contrast, the apparent Vmax for 16 alpha-hydroxylation of dehydroepiandrosterone 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 K(m) for 6 beta-hydroxylations of testosterone and cortisol were greater than those for 16 alpha-hydroxylations (120 and 860 versus 46-58 microM). Thus, CYP3A7 was active for steroid 6 beta-hydroxylations and 16 alpha-hydroxylations, but there were greater differences in Vmax/K(m) ratios between these reactions.
Ohmori
S
S
Division of Pharmacy, Chiba University Hospital, Japan.
Fujiki
N
N
Nakasa
H
H
Nakamura
H
H
Ishii
I
I
Itahashi
K
K
Kitada
M
M
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D016367:Baculoviridae / Q000235:genetics*; D065727:Cytochrome P-450 CYP2C8; D065729:Cytochrome P-450 CYP2C9; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D003687:Dehydroepiandrosterone / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D006854:Hydrocortisone / Q000378:metabolism; D006900:Hydroxylation; D007313:Insecta / Q000378:metabolism*; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D009251:NADPH-Ferrihemoprotein Reductase / Q000096:biosynthesis / Q000378:metabolism*; D014176:Protein Biosynthesis; D039181:Steroid 16-alpha-Hydroxylase; D013256:Steroids / Q000378:metabolism*; D013739:Testosterone / Q000378:metabolism
D013256:Steroids; D013739:Testosterone; D003687:Dehydroepiandrosterone; D003577:Cytochrome P-450 Enzyme System; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; C450259:CYP2C8 protein, human; C103123:CYP3A7 protein, human; D065727:Cytochrome P-450 CYP2C8; D051544:Cytochrome P-450 CYP3A; D039181:Steroid 16-alpha-Hydroxylase; D009251:NADPH-Ferrihemoprotein Reductase; D006854:Hydrocortisone