Identification and characterization of a bacterial cytochrome P450 for the metabolism of diclofenac.
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.
Prior
Jamie E
JE
Department of Chemical and Biological Engineering, University of Colorado, Boulder, CO 80309, USA.
Shokati
Touraj
T
Christians
Uwe
U
Gill
Ryan T
RT
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
D001426:Bacterial Proteins; D004008:Diclofenac; D003577:Cytochrome P-450 Enzyme System