HIGH-YIELD EXPRESSION OF FUNCTIONALLY ACTIVE HUMAN LIVER CYP2D6 IN YEAST-CELLS

In order to develop a model system for studying drug metabolism, we constructed recombinant yeast strains expressing human liver cytochromes P450. A high yield of cDNA-derived CYP2D6 was obtained, due to optimization of the initiation ATG codon context, The PCR-based site-mutagenesis method was used to introduce an AAA sequence immediately before the initiation codon resulting in increased translation of the GAL10-CYCl-derived mRNA. The use of a peptidase-deficient yeast strain also helped to increase the CYP2D6 content, A P450 content of 250 +/- 30 pmol per mg of microsomal protein was achieved, HPLC analysis confirmed that heterologously expressed CYP2D6 catalysed the oxidation of debrisoquine and dextromethorphan, two prototype substrates for CYP2D6. The K-m for debrisoquine 4-hydroxylase was found to be 50 mu M and V-max 7.5 pmol mg(-1) min(-1). Dextromethorphan O-demethylase activity in CYP2D6-containing microsomes was characterized by K-m 8.5 mu M and V-max 700 pmol mg(-1) min(-1). Biotransformation of debrisoquine and dextromethorphan was not detected in control microsomes. Yeast synthesizing CPP2D6 represents a useful in vitro system for studying xenobiotic metabolism.