Constitutive and inducible expression of human cytochrome P450IA1 in yeast Saccharomyces cerevisiae: an alternative enzyme source for in vitro studies

Biochem Biophys Res Commun. 1990 Oct 30;172(2):737-44. doi: 10.1016/0006-291x(90)90736-7.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Benzoflavones / pharmacology
  • Cloning, Molecular
  • Cytochrome P-450 Enzyme System / genetics*
  • Cytochrome P-450 Enzyme System / metabolism
  • Escherichia coli / genetics
  • Gene Library
  • Humans
  • Ketoconazole / pharmacology
  • Microsomes / enzymology
  • Plasmids
  • Recombinant Proteins / metabolism
  • Saccharomyces cerevisiae / genetics*
  • Substrate Specificity

Substances

  • Benzoflavones
  • Recombinant Proteins
  • alpha-naphthoflavone
  • Cytochrome P-450 Enzyme System
  • Ketoconazole