title: Coexpression of a human P450 (CYP3A4) and P450 reductase generates a highly functional monooxygenase system in Escherichia coli.
abstract: The catalytic activities of recombinant cytochrome P450s expressed in E. coli have been impeded by the absence of endogenous P450 reductase. To solve this problem, we coexpressed P450 reductase with CYP3A4. Membranes from this strain contained 215 pmol P450/mg protein and a reductase activity of 1315 nmol cytochrome c reduced/min per mg. We detected 6beta-hydroxylation of testosterone and oxidation of nifedipine in vivo with turnover numbers of 15.2 and 17.3 min(-1), respectively. These values compare favourably with those obtained using an optimally reconstituted system. Our data demonstrate that a catalytically efficient human P450 system can be generated in E. coli.
authors.affiliation: Biomedical Research Centre, Ninewells Hospital and Medical School, Dundee, UK.
authors.forename: J A
authors.initials: JA
authors.lastname: Blake
authors.forename: M
authors.initials: M
authors.lastname: Pritchard
authors.forename: S
authors.initials: S
authors.lastname: Ding
authors.forename: G C
authors.initials: GC
authors.lastname: Smith
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: D001483:Base Sequence; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D006900:Hydroxylation; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase; D009543:Nifedipine / Q000378:metabolism; D010084:Oxidation-Reduction; D010957:Plasmids; D011994:Recombinant Proteins / Q000378:metabolism; D013739:Testosterone / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D013739:Testosterone; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D009247:NADH, NADPH Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase; D009543:Nifedipine