Roles of NADPH-P450 reductase in the O-deethylation of 7-ethoxycoumarin byrecombinant human cytochrome P4501B1 variants in Escherichia coli

Four human cytochrome P450 1B1 (CYP1B1) allelic variants were purified from membranes of Escherichia coli in which respective CYP1B1 cDNAs and human N ADPH-P450 reductase cDNA have been introduced. Purified CYP1B1 variants wer e used to reconstitute 7-ethoxycoumarin O-deethylation activities with puri fied rabbit liver or recombinant (rat) NADPH-P450 reductase in the phosphol ipid vesicles and compared with those catalyzed by CYP1B1 enzymes in the me mbranes of E. coli in monocistronic (by adding the reductase) and bicistron ic (without addition of extra reductase) systems. In the bicistronic system , the ratio of expression of NADPH-P450 reductase to CYP1B1 proteins was fo und to range from 0.2 to 0.5. Purified CYP1B1 enzymes (under optimal recons titution conditions) catalyzed 7-ethoxycoumarin O-deethylation at rates one -third to one-fourth of those catalyzed by membranes of E. coli coexpressin g CYP1B1 and the reductase proteins. Full catalytic activities in reconstit uted systems were achieved with a twofold molar excess of NADPH-P450 reduct ase to CYP1B1; in membranes of E. coli with the monocistronic CYP1B1 constr uct, an eightfold molar excess of reductase to CYP1B1 was required. However , in membranes of bicistronic constructs, there was no additional stimulati on of 7-ethoxycoumarin O-deethylation by extra NADPH-P450 reductase, despit e the fact that the molar ratio of expression levels of reductase to CYP1B1 was <0.5. These results suggest that NADPH-P450 reductase produced in the bacterial membranes is more active in interacting with CYP1B1 proteins in t he bicistronic system than the reductase added to artificial phospholipid v esicles or bacterial membranes, (C) 2000 Academic Press.