title: Functional co-expression of CYP2D6 and human NADPH-cytochrome P450 reductase in Escherichia coli.
abstract: The polymorphic human CYP2D6 has been co-expressed with human NADPH-cytochrome P450 oxidoreductase in Escherichia coli in order to generate a functional recombinant monooxygenase system for the study of xenobiotic metabolism. The two cDNAs were co-expressed from separate, compatible plasmids with different antibiotic selection markers. The CYP2D6 could be detected in bacterial cells at levels up to 700 nmol I-1 culture by Fe(2+)-CO versus Fe2+ difference spectroscopy, exhibiting the characteristic absorbance peak at 450 nm. Immunoblotting demonstrated the presence of both proteins in bacterial membranes, where they were expressed at levels significantly higher than those found in human liver microsomes. Membrane content was 150-200 pmol CYP2D6 (determined spectrally) and 100-230 pmol CYP-reductase (determined enzymatically) per mg protein. Critically, the two co-expressed proteins were able to couple to form a NADPH-dependent monooxygenase which metabolized the CYP2D6 substrate bufuralol (Vmax 3.30 nmol min-1 mg-1 protein; K(m) 11.1 microM) in isolated membrane fractions. This K(m) value was similar to the K(m) determined in human liver microsomes. Activity could be inhibited by the specific inhibitor quinidine. Of greater significance however, was the finding that intact E. coli cells, even in the absence of exogenous NADPH, were able to metabolize bufuralol at rates almost as high as those measured in membranes (4.6 +/- 0.4 min-1 versus 5.7 +/- 0.2 min-1 at 50 microM substrate). Such recombinant strains will greatly facilitate the molecular characterization of allelic variants of cytochrome P450 isoenzymes.
authors.affiliation: Biomedical Research Centre, University of Dundee, Ninewells Hospital and Medical School, UK.
authors.forename: M P
authors.initials: MP
authors.lastname: Pritchard
authors.forename: M J
authors.initials: MJ
authors.lastname: Glancey
authors.forename: J A
authors.initials: JA
authors.lastname: Blake
authors.forename: D E
authors.initials: DE
authors.lastname: Gilham
authors.forename: B
authors.initials: B
authors.lastname: Burchell
authors.forename: C R
authors.initials: CR
authors.lastname: Wolf
authors.forename: T
authors.initials: T
authors.lastname: Friedberg
mesh_terms: D000319:Adrenergic beta-Antagonists / Q000378:metabolism; D001483:Base Sequence; D019389:Cytochrome P-450 CYP2D6 / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D004983:Ethanolamines / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008790:Metoprolol / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011110:Polymorphism, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000319:Adrenergic beta-Antagonists; D017931:DNA Primers; D004983:Ethanolamines; D011994:Recombinant Proteins; C010831:bufuralol; D019389:Cytochrome P-450 CYP2D6; D009251:NADPH-Ferrihemoprotein Reductase; D008790:Metoprolol