Catalytic activities of human debrisoquine 4-hydroxylase cytochrome P450 (CYP2D6) expressed in yeast

Abstract

A 1.57kb BamH1 fragment containing a full-length human debrisoquine 4-hydroxylase cytochrome P450 ( CYP2D6) cDNA was inserted into the BglII site of the yeast expression plasmid pMA91 and the resulting recombinant plasmid, pELT1, introduced into Saccharomyces cerevisiae strain AH22. Microsomes prepared from AH22/pELT1 cells gave an absorption maximum at 448 nm and a P450 content of 67 ± 31 pmol/mg of microsomal protein. No P450 was detectable in microsomes prepared from AH22/pMA91 control cells. A western blot of microsomes prepared from yeast transformed with pELT1 were probed with a monoclonal antibody to CYP2D6 and revealed a strong band with a molecular mass consistent with that of CYP2D6 from human liver microsomes. No corresponding band was observed with microsomes from control yeast transformed with pMA91 alone. Microsomes from AH22/pELT cells showed catalytic activity towards metoprolol (α-hydroxylation and O-demethylation, 0.17 and 0.78 nmol/mg protein/h, respectively); and towards sparteine (2-and 5-dehydrogenation, 1.82 and 0.59 nmol/mg protein/h, respectively). The inhibition of metoprolol metabolism by quinidine (Qd) was 200 times more potent than that of quinine (Qn), both for α-hydroxylation (Qd IC 50) = 0.05 μM; Qn IC 50 = 4 μM) and O-demethylation (Qd IC 50 = 0.05 μM; Qn IC 50 = 4 μM). Negligible metabolism of tolbutamide and S-mephenytoin, substrates of the 2C sub-family, and of p-nitrophenol, a substrate of CYP2E1, was detected, although a trace of the N-deethylated metabolite of lignocaine, thought to be metabolised by CYP3A4, was detected with microsomes from CYP2D6-expressing yeast cells. The results indicate that yeast cells containing human CYP2D6 cDNA express a functionally active form of the enzyme, the immunochemical and catalytic properties of which are consistent with those of human liver.