title: Identification and characterization of a bacterial cytochrome P450 for the metabolism of diclofenac.
abstract: The bacterium Actinoplanes sp. ATCC 53771 is known to perform drug metabolism of several xenobiotics similarly to humans. We identified a cytochrome P450 enzyme from this strain, CYP107E4, and expressed it in Escherichia coli using the pET101 vector. The purified enzyme showed the characteristic reduced-CO difference spectra with a peak at 450 nm, indicating the protein is produced in the active form with proper heme incorporation. The CYP107E4 enzyme was found to bind the drug diclofenac. Using redox enzymes from spinach, the reconstituted system is able to produce hydroxylated metabolites of diclofenac. Production of the human 4'-hydroxydiclofenac metabolite by CYP107E4 was confirmed, and a second hydroxylated metabolite was also produced.
authors.affiliation: Department of Chemical and Biological Engineering, University of Colorado, Boulder, CO 80309, USA.
authors.forename: Jamie E
authors.initials: JE
authors.lastname: Prior
authors.forename: Touraj
authors.initials: T
authors.lastname: Shokati
authors.forename: Uwe
authors.initials: U
authors.lastname: Christians
authors.forename: Ryan T
authors.initials: RT
authors.lastname: Gill
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004008:Diclofenac / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D008848:Micromonosporaceae / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013057:Spectrum Analysis; D018724:Spinacia oleracea / Q000201:enzymology
chemical_list: D001426:Bacterial Proteins; D004008:Diclofenac; D003577:Cytochrome P-450 Enzyme System