Expression, Purification, and Characterization of Mouse Cyp2d22

Abstract

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 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 M) than that (4.2 M) exhibited by purified human CYP2D6. The K m for dextromethorphan N-demethylation by Cyp2d22 was found to be 418 M, 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 M 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 Oor N-demethylation. These results suggest that mouse Cyp2d22 has its own substrate specificity beyond CYP2D6-like-deficient activity.

Functional Expression and Comparative Characterization of Nine Murine Cytochromes P450 by Fluorescent Inhibition Screening

“…Surprisingly, the CYP3A4 specific substrate VIVID CYP3A4 Red proved to be a suitable probe for Cyp2d22, whereas the CYP2D6-specific probes VIVID CYP2D6 Cyan and AMMC were not metabolized. This "un-CYP2D6-like" behavior is supported by work carried out by Yu and Haining (2006), who suggest that Cyp2d22 is not the murine enzyme responsible for CYP2D6-like activity, behaving instead with a more "CYP3A4-like" tendency.…”

Hepatic Cyp2d and Cyp26a1 mRNAs and Activities Are Increased During Mouse Pregnancy

“…Nevertheless, it is not possible to differentiate the relative contributions of individual Cyp2d enzymes to dextrorphan formation. Using Cyp2d knock-out mice, it has been shown that Cyp2d enzymes mediate the overall elimination of dextromethorphan in mice (Scheer et al, 2012), but dextromethorphan metabolism has been kinetically characterized only for Cyp2d22 (K m = 250 mM) (Yu and Haining, 2006). The observed K m values for dextrorphan formation in the mouse liver homogenates (2.5-3.5 mM), were ;40-fold lower than that shown for Cyp2d22, suggesting that multiple enzymes contribute to dextrorphan formation in mouse liver.…”

Expression and Functional Analysis of CYP2D6.24, CYP2D6.26, CYP2D6.27, and CYP2D7 Isozymes

“…The Alliance HPLC system was controlled with Millennium 32 software. A 4.6 ϫ 250 mm, 5 mm zorbax phenyl column (Agilent Technologies, Santa Clara, CA) was used to separate the substrates and the metabolites, similar to the method described previously (Yu et al, 2002;Yu and Haining, 2006). DXM, dextrorphan, and 3-methoxymorphinan were eluted at 22.0, 10.6, and 17.3 min, respectively.…”

“…Incubations were carried out in 100 mM potassium phosphate buffer, pH 7.4, containing 0.1 M individual microsomal CYP2D6 or mitochondrial CYP2D7 isozymes, 0.2 M P450 reductase, 1 mM NADPH, and 200 l of substrate in a final volume, as described previously (Yu et al, 2002;Yu and Haining, 2006). Reactions were initiated by the addition of NADPH.…”