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

Yusuf Y. Deeni, Mark J. I. Paine, Andrew D. Ayrton, Stephen E. Clarke, Richard Chenery, C. Roland Wolf

Research output: Contribution to journalArticle

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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.
Original languageEnglish
Pages (from-to)16-24
Number of pages9
JournalArchives of Biochemistry and Biophysics
Volume396
Issue number1
DOIs
StatePublished - 1 Dec 2001

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NADPH-Ferrihemoprotein Reductase
Cytochrome P-450 CYP2D6
Cytochrome P-450 Enzyme System
Enzymes
Proteins
Fusion reactions
Hydroxylation
Leishmania
Dextromethorphan
Metoprolol
Mixed Function Oxygenases
Oxidoreductases
Escherichia coli
Frangula
Bronchiolo-Alveolar Adenocarcinoma
Cytochrome P-450 CYP3A
Membrane Fusion
Cardiopulmonary Resuscitation
Ion Exchange Chromatography
Affinity Chromatography

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Deeni, Yusuf Y.; Paine, Mark J. I.; Ayrton, Andrew D.; Clarke, Stephen E.; Chenery, Richard; Wolf, C. Roland / Expression, purification, and biochemical characterization of a human cytochrome P450 CYP2D6-NADPH cytochrome P450 reductase fusion protein.

In: Archives of Biochemistry and Biophysics, Vol. 396, No. 1, 01.12.2001, p. 16-24.

Research output: Contribution to journalArticle

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Expression, purification, and biochemical characterization of a human cytochrome P450 CYP2D6-NADPH cytochrome P450 reductase fusion protein. / Deeni, Yusuf Y.; Paine, Mark J. I.; Ayrton, Andrew D.; Clarke, Stephen E.; Chenery, Richard; Wolf, C. Roland.

In: Archives of Biochemistry and Biophysics, Vol. 396, No. 1, 01.12.2001, p. 16-24.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Expression, purification, and biochemical characterization of a human cytochrome P450 CYP2D6-NADPH cytochrome P450 reductase fusion protein

AU - Deeni,Yusuf Y.

AU - Paine,Mark J. I.

AU - Ayrton,Andrew D.

AU - Clarke,Stephen E.

AU - Chenery,Richard

AU - Wolf,C. Roland

PY - 2001/12/1

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N2 - 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.

AB - 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.

U2 - 10.1006/abbi.2001.2585

DO - 10.1006/abbi.2001.2585

M3 - Article

VL - 396

SP - 16

EP - 24

JO - Archives of Biochemistry and Biophysics

T2 - Archives of Biochemistry and Biophysics

JF - Archives of Biochemistry and Biophysics

SN - 0003-9861

IS - 1

ER -