Expression in Saccharomyces cerevisiae of chimeric cytochrome P450 cDNAs constructed from cDNAs for rat cytochrome P450c and P450d.
Three chimeric cytochrome P450 cDNAs were constructed by replacing the central region, carboxy-terminal region, or both central and carboxy-terminal regions of cytochrome P450c cDNA with the corresponding regions of cytochrome P450d cDNA. These were inserted between the alcohol dehydrogenase I promoter and terminator of yeast expression vector pAAH5 to form expression plasmids pACDC2, pACCD1, and pACDD2. On introduction of each of these plasmids into Saccharomyces cerevisiae AH22 cells, chimeric cytochrome P450 proteins were expressed in AH22/pACDC2, AH22/pACCD1, and AH22/pACDD2 cells at the level of at least 10(5), 4 X 10(5) molecules per cell, respectively. The reduced CO-difference spectra showed that AH22/pACCD1 and AH22/pACDD2 cells contained 4 X 10(5) and 10(5) molecules per cell of the corresponding chimeric cytochrome P450 hemoproteins, designated as cytochrome P450ccd and cytochrome P450cdd, respectively. Cytochrome P450ccd exhibited higher monooxygenase activities toward 7-ethoxycoumarin, acetanilide, and benzo[alpha]pyrene than cytochrome P450c, although the substrate specificity of cytochrome P450ccd seemed to be the same as that of cytochrome P450c. Cytochrome P450cdd exhibited lower activities toward 7-ethoxycoumarin and benzo[alpha]pyrene, and a higher activity toward acetanilide as compared with those of cytochrome P450c and cytochrome P450ccd. Therefore, the substrate specificity of cytochrome P450cdd seemed to be the same as that of cytochrome P450d. These results suggest that the central one-third region of cytochrome P450c and cytochrome P450d is responsible for substrate-binding, and that the carboxy-terminal third of both cytochromes P450 plays an important role in electron transport.
D000818:Animals; D001665:Binding Sites; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D004247:DNA / Q000235:genetics; D004274:DNA, Recombinant; D005420:Flavoproteins / Q000378:metabolism; D008861:Microsomes / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000378:metabolism; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013329:Structure-Activity Relationship
D004274:DNA, Recombinant; D005420:Flavoproteins; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases