FUNCTIONAL EXPRESSION OF FUSED ENZYMES BETWEEN HUMAN CYTOCHROME P4501A1 AND HUMAN NADPH-CYTOCHROME P450 OXIDOREDUCTASE IN SACCHAROMYCES-CEREVISIAE

The activity of human cytochrome P450 enzymes heterologously expressed in Saccharomyces cerevisiae cells is limited by the yeast endogenous cytochrome P450 oxidoreductase (yOR), To overcome these limitations, w e constructed hybrids between human P4501A1 (CYP1A1) and human P450 ox idoreductase (hOR) by combining the cDNA encoding hOR with the CYP1A1 cDNA, In addition, in one construct, the amino terminus of hOR was rep laced by the membrane anchor domain of a yeast protein. Anchoring of t he fusion constructs in internal membranes either by the amino terminu s of hOR or by the yeast peptide resulted in functional hybrid protein s, which were present in similar amounts as the authentic CYP1A1 in mi crosomal fractions of recombinant cells, Saccharomyces cerevisiae cell s transformed with the expression plasmids produced the respective pro teins in the expected molecular sizes reactive with both anti-CYP1A im munoglobulin (Ig) and anti-oxidoreductase Ig. Saccharomyces cerevisiae yOR-mutant (cprl(-)) and wild-type (CPRl(+)) cells containing the fus ed enzymes exhibited GYP1A1-specific 7-ethoxyresorufin-O-deethylase ac tivities, Reduced GO-difference spectra of microsomal fractions contai ning the fused enzymes indicated a proper incorporation of protoheme i nto the GYP1A1 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 r eductases and able to activate the yeast P450 enzyme lanosterol-14 alp ha-demethylase, indicating correct insertion of the chimeric proteins in internal membranes.