title: Functional expression of fused enzymes between human cytochrome P4501A1 and human NADPH-cytochrome P450 oxidoreductase in Saccharomyces cerevisiae.
abstract: The activity of human cytochrome P450 enzymes heterologously expressed in Saccaromyces cerevisiae cells is limited by the yeast endogenous cytochrome P450 oxidoreductase (yOR). To overcome these limitations, we constructed hybrids between human P4501A1 (CYP1A1) and human P450 oxidoreductase (hOR) by combining the cDNA encoding hOR with the CYP1A1 cDNA. In addition, in one construct, the amino terminus of hOR was replaced by the membrane anchor domain of a yeast protein. Anchoring of the fusion constructs in internal membranes either by the amino terminus of hOR or by the yeast peptide resulted in functional hybrid proteins, which were present in similar amounts as the authentic CYP1A1 in microsomal fractions of recombinant cells. Saccharomyces cerevisiae cells transformed with the expression plasmids produced the respective proteins in the expected molecular sizes reactive with both anti-CYP1A immunoglobulin (Ig) and anti-oxidoreductase Ig. Saccharomyces cerevisiae yOR-mutant (cpr1-) and wild-type (CPR1+) cells containing the fused enzymes exhibited CYP1A1-specific 7-ethoxyresorufin-O-deethylase activities. Reduced CO-difference spectra of microsomal fractions containing the fused enzymes indicated a proper incorporation of protoheme into the CYP1A1 domains. These results show that the chimeric proteins represent catalytically self-sufficient monooxygenase systems. The hOR domains of the hybrid proteins were also functional as cytochrome c reductases and able to activate the yeast P450 enzyme lanosterol-14 alpha-demethylase, indicating correct insertion of the chimeric proteins in internal membranes.
authors.affiliation: Institute of Toxicology, Swiss Federal Institute of Technology, Schwerzenbach.
authors.forename: N E
authors.initials: NE
authors.lastname: Wittekindt
authors.forename: F E
authors.initials: FE
authors.lastname: Würgler
authors.forename: C
authors.initials: C
authors.lastname: Sengstag
mesh_terms: D003001:Cloning, Molecular; D019363:Cytochrome P-450 CYP1A1; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D004352:Drug Resistance, Microbial / Q000235:genetics; D006801:Humans; D007654:Ketoconazole / Q000494:pharmacology; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics* / Q000378:metabolism; D010088:Oxidoreductases / Q000378:metabolism; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics*
chemical_list: D011993:Recombinant Fusion Proteins; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D019363:Cytochrome P-450 CYP1A1; D009251:NADPH-Ferrihemoprotein Reductase; D007654:Ketoconazole