Expression, purification, and characterization of mouse CYP2d22.
Metabolism of the prototype human CYP2D6 substrates debrisoquine and bufuralol proceeds at a much slower rate in mice; therefore, the mouse has been proposed as an animal model for the human CYP2D6 genetic deficiency. To interpret the molecular mechanism of this deficiency, a cDNA belonging to the CYP2D gene subfamily (Cyp2d22) has been cloned and sequenced from a mouse mammary tumor-derived cell line. In the current study, Cyp2d22 enzyme was overexpressed and purified from insect cells using a baculovirus-mediated system. The activity of this purified enzyme was directly compared with purified human CYP2D6 toward codeine, dextromethorphan, and methadone as substrates. Purified Cyp2d22 was found to catalyze the O-demethylation of dextromethorphan with significantly higher K(m) values (250 microM) than that (4.2 microM) exhibited by purified human CYP2D6. The K(m) for dextromethorphan N-demethylation by Cyp2d22 was found to be 418 microM, much lower than that observed with human CYP2D6 and near the K(m) for dextromethorphan N-demethylation catalyzed by CYP3A4. CYP2D6 catalyzed codeine O-demethylation, whereas Cyp2d22 and CYP3A4 mediated codeine N-demethylation. Furthermore, methadone, a known CYP3A4 substrate and CYP2D6 inhibitor, was N-demethylated by Cyp2d22 with a K(m) of 517 microM and V(max) of 4.9 pmol/pmol/min. Quinidine and ketoconazole, potent inhibitors to CYP2D6 and CYP3A4, respectively, did not show strong inhibition toward Cyp2d22-mediated dextromethorphan O- or N-demethylation. These results suggest that mouse Cyp2d22 has its own substrate specificity beyond CYP2D6-like-deficient activity.
Yu
Ai-Ming
AM
Department of Basic Pharmaceutical Sciences, School of Pharmacy, West Virginia University, Morgantown, WV 26506-9530, USA.
Haining
Robert L
RL
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae; D002460:Cell Line; D003001:Cloning, Molecular; D003061:Codeine / Q000378:metabolism*; D019389:Cytochrome P-450 CYP2D6 / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D065690:Cytochrome P-450 CYP2D6 Inhibitors; D051544:Cytochrome P-450 CYP3A; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000072467:Cytochrome P450 Family 2; D003640:Dealkylation; D003915:Dextromethorphan / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005473:Fluoxetine / Q000378:metabolism*; D007313:Insecta; D007654:Ketoconazole / Q000494:pharmacology; D007700:Kinetics; D008691:Methadone / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011802:Quinidine / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D065690:Cytochrome P-450 CYP2D6 Inhibitors; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D005473:Fluoxetine; D003915:Dextromethorphan; D003577:Cytochrome P-450 Enzyme System; C414739:Cyp2d22 protein, mouse; D019389:Cytochrome P-450 CYP2D6; D051544:Cytochrome P-450 CYP3A; D000072467:Cytochrome P450 Family 2; C510163:CYP3A4 protein, human; D011802:Quinidine; D007654:Ketoconazole; D008691:Methadone; D003061:Codeine