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Please use this identifier to cite or link to this item: http://hdl.handle.net/10373/229

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Title: Expression, purification, and biochemical characterization of a human cytochrome P450 CYP2D6-NADPH cytochrome P450 reductase fusion protein
Authors: Deeni, Yusuf Y.
Paine, Mark J. I.
Ayrton, Andrew D.
Clarke, Stephen E.
Chenery, Richard
Wolf, C. Roland
Affiliation: University of Abertay Dundee. School of Social & Health Sciences
Keywords: CYP2D6
Drug metabolism
Cytochrome P450
NADPH cytochrome P450 reductase
Fusion
Purification
Issue Date: 1-Dec-2001
Publisher: Elsevier
Type: Journal Article
Refereed: peer-reviewed
Rights: Published version (c)Elsevier, available from: DOI: 10.1006/abbi.2001.2585
Citation: Deeni, Y. Y., et al. Expression, purification, and biochemical characterization of a human cytochrome P450 CYP2D6-NADPH cytochrome P450 reductase fusion protein. Archives of Biochemistry and Biophysics. 396(1): pp.16-24. [Online] Available from: DOI: 10.1006/abbi.2001.2585
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.
URI: http://hdl.handle.net/10373/229
ISSN: 0003-9861
Appears in Collections:Social & Health Sciences Collection

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