In order to develop a model system for studying drug metabolism, we co
nstructed recombinant yeast strains expressing human liver cytochromes
P450. A high yield of cDNA-derived CYP2D6 was obtained, due to optimi
zation of the initiation ATG codon context, The PCR-based site-mutagen
esis method was used to introduce an AAA sequence immediately before t
he initiation codon resulting in increased translation of the GAL10-CY
Cl-derived mRNA. The use of a peptidase-deficient yeast strain also he
lped to increase the CYP2D6 content, A P450 content of 250 +/- 30 pmol
per mg of microsomal protein was achieved, HPLC analysis confirmed th
at heterologously expressed CYP2D6 catalysed the oxidation of debrisoq
uine 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 CYP2D
6-containing microsomes was characterized by K-m 8.5 mu M and V-max 70
0 pmol mg(-1) min(-1). Biotransformation of debrisoquine and dextromet
horphan was not detected in control microsomes. Yeast synthesizing CPP
2D6 represents a useful in vitro system for studying xenobiotic metabo
lism.