Received for publication December 12, 2005.
Revised March 28, 2006.
Accepted for publication March 29, 2006.

Expression, Purification and Characterization of Mouse Cyp2d22

Abstract

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 over-expressed 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 µM) than that (4.2 µM) exhibited by purified human CYP2D6. The K_m for dextromethorphan N-demethylation by Cyp2d22 was found to be 418 µM, 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 µM 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.

Key words: CYP2D, CYP3A, enzyme inhibitors, enzyme kinetics, human CYP enzymes, metabolite kinetics

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