Functional Expression of Fused Enzymes Between Human Cytochrome P4501A1 and Human NADPH-Cytochrome P450 Oxidoreductase inSaccharomyces cerevisiae

Abstract

The activity of human cytochrome P450 enzymes heterologously expressed in Saccaromyces cerevisiae cells is limited by the yeast endogenous cytochrome P450 oxidoreductase (yOR). To overcome these limitations, we constructed hybrids between human P4501A1 (CYP1A1) and human P450 oxidoreductase (hOR) by combining the cDNA encoding hOR with the CYP1A1 cDNA. In addition, in one construct, the amino terminus of hOR was replaced by the membrane anchor domain of a yeast protein. Anchoring of the fusion constructs in internal membranes either by the amino terminus of hOR or by the yeast peptide resulted in functional hybrid proteins, which were present in similar amounts as the authentic CYP1A1 in microsomal fractions of recombinant cells. Saccharomyces cerevisiae cells transformed with the expression plasmids produced the respective proteins in the expected molecular sizes reactive with both anti-CYP1A immunoglobulin (Ig) and anti-oxidoreductase Ig. Saccharomyces cerevisiae yOR-mutant (cpr1-) and wild-type (CPR1+) cells containing the fused enzymes exhibited CYP1A1-specific 7-ethoxyresorufin-O-deethylase activities. Reduced CO-difference spectra of microsomal fractions containing the fused enzymes indicated a proper incorporation of protoheme into the CYP1A1 domains. These results show that the chimeric proteins represent catalytically self-sufficient monooxygenase systems. The hOR domains of the hybrid proteins were also functional as cytochrome c reductases and able to activate the yeast P450 enzyme lanosterol-14 alpha-demethylase, indicating correct insertion of the chimeric proteins in internal membranes.

Metabolism of phenanthrene by house fly CYP6D1 and dog liver cytochrome P450

“…However, overexpression of yeast P450 reductase enhances activity of heterologously expressed P450s [13]. Furthermore, protein fusion between P450s and P450 reductase has resulted in P450 activity [35,40]. Although heterologous expression of well characterized P450s that metabolize PAHs holds promise as a potential tool in metabolism and bioremediation of PAH contamination, the above mentioned biological and technical limitations must first be overcome.…”

Structural domains of P450-containing monooxygenase systems

“…Reciprocally, the possibility of obtaining a functional one-component P450 system from a two-component system has been shown in gene fusion experiments. To date, the following eukaryotic analogues of P450 BM-3 have been constructed: bovine CYP17/yeast CPR (Shibata et al, 1990), rat CYP1A1/yeast CPR (Sakaki et al, 1994) and human CYP1A1/human CPR (Wittekindt et al, 1995) expressed in Saccharomyces cerevisiae; bovine CYP17/rat CPR, rat CYP4A1/rat CPR (Fisher et al, 1992;Shet et al, 1994) and human CYP3A4/ human or rat CPR (Shet et al, 1993) expressed in E. coli.…”

A wide-range integrative yeast expression vector system based on Arxula adeninivorans-derived elements

“…As eukaryotes, they are able to perform complex posttranslational modifications, thus producing foreign proteins that are often identical or very similar to native products of mammalian sources [8,9,11,33,37,39]. The first yeast species to be employed for the production of foreign proteins was Saccharomyces cerevisiae.…”