Constitutive and inducible expression of human cytochrome P450IA1 in yeast Saccharomyces cerevisiae: an alternative enzyme source for in vitro studies.
A cDNA of human cytochrome P450IA1 was expressed in yeast Saccharomyces cerevisiae on a multicopy plasmid under the control of the constitutive GAPFL or the inducible PHO5 promoter. Microsomes of transformed yeast contained substantial amounts of the heterologous enzyme as determined by reduced CO-difference spectra (156-68 pmol/mg). Enzyme kinetics with 7-ethoxyresorufin as substrate resulted in a Km value of 92 nM and a Vmax value of 223 pmol/mg/min, which is comparable to data obtained with human liver microsomes. The antimycotic drug ketoconazole (Ki = 22nM) as well as the isozyme specific P450 inhibitor alpha-naphthoflavone (Ki = 1.2 nM) were shown to be strong inhibitors of human P450IA1. Taken together, these data show that heterologous P450 gene expression in yeast is a potent instrument for the study of enzyme specific parameters and might be used to answer further questions with regard to substrate specificity as well as drug interaction in a background with no interfering activities.
Eugster
H P
HP
Institute of Toxicology, Swiss Federal Institute of Technology, Schwerzenbach.
Sengstag
C
C
Meyer
U A
UA
Hinnen
A
A
Würgler
F E
FE
D001571:Benzoflavones / Q000494:pharmacology; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D006801:Humans; D007654:Ketoconazole / Q000494:pharmacology; D008861:Microsomes / Q000201:enzymology; D010957:Plasmids; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013379:Substrate Specificity
D001571:Benzoflavones; D011994:Recombinant Proteins; C011512:alpha-naphthoflavone; D003577:Cytochrome P-450 Enzyme System; D007654:Ketoconazole