A genetically engineered P450 monooxygenase: construction of the functional fused enzyme between rat cytochrome P450c and NADPH-cytochrome P450 reductase.
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 microsomal membrane with the reductase moiety lying on the cytoplasmic side.
D000818:Animals; D003374:Coumarins / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D004247:DNA / Q000235:genetics; D004274:DNA, Recombinant; D005813:Genes, Synthetic; D005822:Genetic Vectors; D007425:Intracellular Membranes / Q000032:analysis; D007700:Kinetics; D008565:Membrane Proteins / Q000032:analysis; D008861:Microsomes / Q000201:enzymology; D008970:Molecular Weight; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000032:analysis / Q000235:genetics
D003374:Coumarins; D004274:DNA, Recombinant; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C017299:7-ethoxycoumarin; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase