Engineered yeast cells as model to study coupling between human xenobiotic metabolizing enzymes. Simulation of the...

Human microsomal epoxide hydrolase and cytochrome P450 (P450) 1A1 were coexpressed in Saccharomyces cerevisiae from expression cassettes integrated respectively into the host chromosomal DNA and on a multicopy plasmid in a strain already overexpressing yeast NADPH-cytochrome P450 reductase (P450 reductase). A styrene-oxide-hydrolase activity (2 nmol.min-1.mg microsomal protein-1) and a 7-ethoxyresorufin-O-deethylase activity (320 pmol.min-1.mg microsomal protein-1) characteristic respectively of microsomal epoxide hydrolase and P450 1A1 were detected. The conversion of benzo[a]pyrene (B[a]P) to B[a]P-7,8-dihydrodiol both in microsomal preparations and in growing yeast cells was observed, demonstrating an efficient coupling between the two human enzymes. Kinetic analysis indicated that the B[a]P-7,8-oxide produced by the P450-1A1-dependent reaction does not accumulate before hydrolysis by microsomal epoxide hydrolase. This system was also used as a control to evaluate the coupling efficiency of a mixture of microsomes or of yeast cells containing separately the individual enzymes (i.e., human P450 1A1 and microsomal epoxide hydrolase). B[a]P-7,8-oxide was well converted to the corresponding dihydrodiol with a mixture of microsomes. In contrast, when the same experiment was repeated with a mixture of cells expressing independently the two activities, dihydrodiol formation was not observed. Coexpression of human phase I and phase II enzymes in a single yeast cell and microsome mixture thus appear to be complementary tools for the simulation of human-drug-metabolism or carcinogen-metabolism pathways.