title: Drug metabolism by Escherichia coli expressing human cytochromes P450.
abstract: The broad substrate specificity of the cytochrome P450 (P450) enzyme superfamily of heme-thiolate proteins lends itself to diverse environmental and pharmaceutical applications. Until recently, the primary drawback in using living bacteria to catalyze mammalian P450-mediated reactions has been the paucity of electron transport from NADPH to P450 via endogenous flavoproteins. We report the functional expression in Escherichia coli of bicistronic constructs consisting of a human microsomal P450 enzyme encoded by the first cistron and the auxiliary protein NADPH-P450 reductase by the second. Expression levels of P450s ranged from 35 nmol per liter culture to 350 nmol per liter culture, with expression of NADPH-P450 reductase typically ranging from 50% to 100% of that of P450. Transformed bacteria metabolized a number of typical P450 substrates at levels comparable to isolated bacterial membranes fortified with an NADPH-generating system. These rates compare favorably with those obtained using human liver microsomes as well as those of reconstituted in vitro systems composed of purified proteins, lipids, and cofactors.
authors.affiliation: Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
authors.forename: A
authors.initials: A
authors.lastname: Parikh
authors.forename: E M
authors.initials: EM
authors.lastname: Gillam
authors.forename: F P
authors.initials: FP
authors.lastname: Guengerich
mesh_terms: D002384:Catalysis; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004926:Escherichia coli; D006801:Humans; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase; D009876:Operon; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis; D013379:Substrate Specificity
chemical_list: D018076:DNA, Complementary; D004364:Pharmaceutical Preparations; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D009247:NADH, NADPH Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase