FUNCTIONAL EXPRESSION OF HOUSE-FLY (MUSCA-DOMESTICA) CYTOCHROME-P450 CYP6D1 IN YEAST (SACCHAROMYCES-CEREVISIAE)

Cytochrome P450 CYP6D1 from the house fly is important in the detoxica tion of xenobiotics and in resistance to pyrethroid insecticides, In h ouse fly microsomes CYP6D1 requires cytochrome b(5) for the metabolism of some substrates, such as benzo[a]pyrene, but does not require cyto chrome b(5) for the metabolism of other substrates such as methoxyreso rufin. To examine the molecular mechanisms involved in its metabolism of pyrethroids and other substrates, a system for the heterologous exp ression of CYP6D1 in the yeast Saccharomyces cerevisiae was developed, Heterologous CYP6D1 can be inducibly expressed by culture in media wi th galactose as the sore carbon source, and is successfully inserted i nto the yeast microsomes, CYP6D1 is enzymatically active, as measured by methoxyresorufin-O-demethylation, indicating that CYP6D1 is able to interact with yeast P450 reductase, However, CYP6D1 expression did no t result in measurable benzo[a]pyrene hydroxylation, suggesting that C YP6D1 cannot interact with yeast cytochrome b(5), or that there is ins ufficient cytochrome b(5) in the yeast microsomes to support this CYP6 D1-mediated activity. Some suggestions are made for improving the yeas t microsomal oxidoreductase environment in order to optimize CYP6D1 fu nction. (C) 1997 Elsevier Science Ltd. All rights reserved.