title: Functional expression of house fly (Musca domestica) cytochrome P450 CYP6D1 in yeast (Saccharomyces cerevisiae).
abstract: Cytochrome P450 CYP6D1 from the house fly is important in the detoxication of xenobiotics and in resistance to pyrethroid insecticides. In house fly microsomes CYP6D1 requires cytochrome b5 for the metabolism of some substrates, such as benzo[a]pyrene, but does not require cytochrome b5 for the metabolism of other substrates such as methoxyresorufin. To examine the molecular mechanisms involved in its metabolism of pyrethroids and other substrates, a system for the heterologous expression of CYP6D1 in the yeast Saccharomyces cerevisiae was developed. Heterologous CYP6D1 can be inducibly expressed by culture in media with galactose as the sole carbon source, and is successfully inserted into 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 not result in measurable benzo[a]pyrene hydroxylation, suggesting that CYP6D1 cannot interact with yeast cytochrome b5, or that there is insufficient cytochrome b5 in the yeast microsomes to support this CYP6D1-mediated activity. Some suggestions are made for improving the yeast microsomal oxidoreductase environment in order to optimize CYP6D1 function.
authors.affiliation: Department of Entomology, Cornell University, Ithaca, NY 14853, USA.
authors.forename: F F
authors.initials: FF
authors.lastname: Smith
authors.forename: J G
authors.initials: JG
authors.lastname: Scott
mesh_terms: D000818:Animals; D001564:Benzo(a)pyrene / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D000072472:Cytochrome P450 Family 6; D015786:Cytochromes b5 / Q000378:metabolism; D005690:Galactose / Q000494:pharmacology; D006793:Houseflies / Q000201:enzymology* / Q000235:genetics; D019476:Insect Proteins; D008861:Microsomes / Q000201:enzymology; D009153:Mutagens / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase; D010088:Oxidoreductases / Q000032:analysis; D012331:RNA, Fungal / Q000032:analysis / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
chemical_list: D019476:Insect Proteins; D009153:Mutagens; D012331:RNA, Fungal; D001564:Benzo(a)pyrene; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; C076307:methoxyresorufin-O-demethylase; C092307:CYP6D1 protein, Musca domestica; D000072472:Cytochrome P450 Family 6; D009247:NADH, NADPH Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase; D005690:Galactose