Expression, purification, and biochemical characterization of a human cytochrome P450 CYP2D6-NADPH cytochrome P450 reductase fusion protein

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 l′-hydroxylation, metoprolol α-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 Km and kcat values for bufuralol metabolism were estimated to be 10.2 μM 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 l′-hydroxylation, metoprolol α-hydroxylation, O-desmethylation, and dextromethorphan O-demethylation were 1.2, 0.52, 0.79, and 0.76 min-1, respectively. Bufuralol l′-hydroxylase activity by purified CYP2D6F was enhanced by phospholipids and added CPR. The CYP2D6F enzyme was able to stimulate CYP3A4 testosterone 6β-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.