Heterologous expression of human microsomal epoxide hydrolase in Saccharomyces cerevisiae. Study of the valpromide-carbamazepine epoxide interaction.
A cDNA of human microsomal epoxide hydrolase (hmEH) was constitutively and inducibly expressed in Saccharomyces cerevisiae. The heterologous enzyme was located mainly in the microsomal fraction of yeast cells. Yeast microsomes containing hmEH exerted styrene oxide hydrolase activity (Km = 300 microM; Vmax = 22 nmol/mg min) as well as carbamazepine epoxide hydrolase activity. The hmEH catalysed exclusively the formation of carbamazepine-10,11-transdihydrodiol, since no carbamazepine-10,11-cisdihydrodiol was detected. Inhibition studies using these microsomes revealed unequivocally hmEH as the target for inhibition by the antiepileptic drug valpromide. A Ki value of 27 microM was determined for the inhibitor valpromide with styrene oxide as substrate. For carbamazepine epoxide, a Ki value of 8.6 microM was obtained, which is well in line with data published for hmEH determined with human liver microsomes. Our results demonstrate the potential of heterologous gene expression in S. cerevisiae and its application to the in vitro study of pharmacological and toxicological problems.
Eugster
H P
HP
Institute of Toxicology, Swiss Federal Institute of Technology, Basel.
Sengstag
C
C
Hinnen
A
A
Meyer
U A
UA
Würgler
F E
FE
D001483:Base Sequence; D002220:Carbamazepine / Q000031:analogs & derivatives / Q000378:metabolism*; D004347:Drug Interactions; D004851:Epoxide Hydrolases / Q000037:antagonists & inhibitors / Q000235:genetics*; D004852:Epoxy Compounds / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007700:Kinetics; D008861:Microsomes / Q000187:drug effects / Q000201:enzymology; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013343:Styrenes / Q000378:metabolism; D014635:Valproic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology
D004852:Epoxy Compounds; D013343:Styrenes; D002220:Carbamazepine; D014635:Valproic Acid; C013690:styrene oxide; D004851:Epoxide Hydrolases; C012227:carbamazepine epoxide; C004651:dipropylacetamide