EXPRESSION OF 4 RAT CYP2D ISOFORMS IN SACCHAROMYCES-CEREVISIAE AND THEIR CATALYTIC SPECIFICITY

We cloned four cDNAs belonging to the CYP2D subfamily to express these enzymes in yeast cells and to compare their catalytic activities simu ltaneously. Three are believed to be alleles of CYP2D1, 2D2, and 2D3, respectively, based on high nucleotide sequence similarity, while CYP2 D4 had both sequences of CYP2D4 and CYP2D18. Expression plasmids carry ing CYP2D cDNAs were transformed into Saccharomyces cerevisiae. Typica l P450 GO-difference spectra with absorbance maximum at 448 nm were re corded with microsomal preparations from the yeast cells expressing th e four CYP2D forms. A catalytic study of these CYP2D forms was done wi th debrisoquine, bufuralol, and lidocaine. CYP2D2 had the highest debr isoquine 4-hydroxylation (2.2 nmol/min/nmol P450) activity, similar to that (2.2 nmol/min/nmol) of human CYP2D6 expressed in yeast cells. CY P2D3 had high lidocaine N-deethylation (43 nmol/min/nmol P450) activit y, and both CYP2D3 and 2D2 exhibited high lidocaine 3-hydroxylation (2 .4 and 1.6 nmol/min/nmol P450, respectively) activity. Bufuralol 1'-hy droxylation catalytic capabilities were comparable among the four isof orms. The activity of CYP2D1 was relatively low toward the three subst rates (debrisoquine, 0.091; bufuralol, 1.5; Lidocaine S-hydroxylation, 0.019; lidocaine N-deethylation, 2.8 nmol/min/nmol P450). These findi ngs indicate that debrisoquine, a typical substrate for CYP2D forms, w as mainly metabolized by CYP2D2 but not CYP2D1 in rat liver and that t he CYP2D forms have different substrate specificity. (C) 1997 Academic Press.