A Genetically Engineered P450 Monooxygenase: Construction of the Functional Fused Enzyme between Rat Cytochrome P450c and NADPH-Cytochrome P450 Reductase

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Abstract
A hybrid cDNA encoding a fused enzyme consisting of rat cytochrome P450c and rat NADPH-cytochrome P450 reductase was constructed by combining the cytochrome P450c cDNA with the cDNA fragment encoding the protease-solubilized moiety of the NADPH-cytochrome P450 reductase. The hybrid cDNA was inserted between the yeast alcohol dehydrogenase I promoter and terminator of the expression vector pAAH5 to yield expression plasmid pAMP19. Saccharomyces cerevisiae AH22 cells transformed with the expression plasmid pAMP19 produced a 130-kD protein reactive with both anti-cytochrome P450c Ig and antireductase Ig. The yeast cells containing the fused enzyme exhibited about four times higher monooxygenase activity toward 7-ethoxycoumarin than those containing rat cytochrome P450c alone. The fused enzyme was purified from the yeast microsomal fraction by sequential chromatography with DEAE-cellulose and 2′,5′-ADP Sepharose 4B columns. The preparation had an apparent molecular weight of 130 kD and the same sequence of the 10 amino-terminal amino acids as that of rat cytochrome P450c. Spectral properties of the fused enzyme indicated the presence of a protoheme, flavin adenine dinucleotide, and flavin mononucleotide in the molecule. The reaction mechanism of the fused enzyme followed first-order kinetics. These results clearly indicate that the fused enzyme is a new self-catalytic P450 monooxygenase. Trypsin treatment of yeast microsomes containing the fused enzyme suggested that the P450 moiety is embedded in the micro-somal membrane with the reductase moiety lying on the cytoplasmic side.