title: Expression, purification, and biochemical characterization of a human cytochrome P450 CYP2D6-NADPH cytochrome P450 reductase fusion protein.
abstract: Cytochrome P450 CYP2D6 metabolizes a wide range of pharmaceutical compounds. A CYP2D6 fusion enzyme (CYP2D6F), containing an amino-terminal human CYP2D6 sequence and a carboxyterminal human NADPH-cytochrome P450 oxidoreductase (CPR) moiety, was constructed. High levels of expression were achieved in Escherichia coli (60-100 nmol/liter) and the enzyme was catalytically active with optimal activities achieved in the presence of the antioxidant, GSH. Turnover values for bufuralol 1'-hydroxylation, metoprolol alpha-hydroxylation, O-desmethylation, and dextromethorphan O-demethylation, using membranes expressing the fusion enzyme, were 5.6, 0.4, 0.72, and 6.19 min(-1), respectively. These values were similar to E. coli membranes which coexpressed human CYP2D6 and CPR (CYP2D6/R). The K(m) and k(cat) values for bufuralol metabolism were estimated to be 10.2 microM and 4.1 min(-1), respectively. The enzyme was purified using ion-exchange chromatography, affinity chromatography (2'-5' ADP-Sepharose), and gel filtration. Estimated turnover rates for bufuralol 1'-hydroxylation, metoprolol alpha-hydroxylation, O-desmethylation, and dextromethorphan O-demethylation were 1.2, 0.52, 0.79, and 0.76 min(-1), respectively. Bufuralol 1'-hydroxylase activity by purified CYP2D6F was enhanced by phospholipids and added CPR. The CYP2D6F enzyme was able to stimulate CYP3A4 testosterone 6beta-hydroxylase activity in a reconstitution system indicating that electron transfer may be largely intermolecular. The catalytically self-sufficient CYP2D6F enzyme will facilitate investigations of P450-CPR interactions and the development of new biocatalysts.
authors.affiliation: Biomedical Research Centre, University of Dundee, Dundee, DD1 9SY, United Kingdom.
authors.forename: Y Y
authors.initials: YY
authors.lastname: Deeni
authors.forename: M J
authors.initials: MJ
authors.lastname: Paine
authors.forename: A D
authors.initials: AD
authors.lastname: Ayrton
authors.forename: S E
authors.initials: SE
authors.lastname: Clarke
authors.forename: R
authors.initials: R
authors.lastname: Chenery
authors.forename: C R
authors.initials: CR
authors.lastname: Wolf
mesh_terms: D019389:Cytochrome P-450 CYP2D6 / Q000235:genetics / Q000378:metabolism*; D003915:Dextromethorphan / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D004983:Ethanolamines / Q000378:metabolism; D006801:Humans; D008790:Metoprolol / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics / Q000378:metabolism*; D004364:Pharmaceutical Preparations / Q000378:metabolism; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D013678:Technology, Pharmaceutical
chemical_list: D004983:Ethanolamines; D004364:Pharmaceutical Preparations; D011993:Recombinant Fusion Proteins; D003915:Dextromethorphan; C010831:bufuralol; D019389:Cytochrome P-450 CYP2D6; D009251:NADPH-Ferrihemoprotein Reductase; D008790:Metoprolol