Genetically engineered modification of P450 monooxygenases: functional analysis of the amino-terminal hydrophobic region and hinge region of the P450/reductase fused enzyme.
Modified constructions of a microsomal cytochrome P450, of NADPH-cytochrome P450 reductase, and of a P450/reductase fused enzyme were prepared to analyze the function of the amino-terminal hydrophobic regions of these enzymes and the hinge region of the fused enzyme. Expression plasmids for delta P450c, delta reductase, and the delta P450/reductase fused enzyme, all of which lacked their amino-terminal hydrophobic regions, were constructed by inserting each of the corresponding cDNAs between the yeast alcohol dehydrogenase I promoter and the terminator of the expression vector pAAH5. Yeast transformed with plasmids encoding delta P450 and the delta P450/reductase fused enzyme produced smaller amounts of the respective enzymes and showed lower monooxygenase activity toward 7-ethoxycoumarin than did yeast transformed with plasmids encoding the complete enzymes. Both delta P450 and delta P450/reductase were found in the microsomal fraction of the yeast cells. Yeast transformed with the expression plasmid for delta reductase produced 20 times more enzyme than did yeast transformed with the plasmid for the complete enzyme. delta Reductase was present in the soluble fraction and was 33 times more active in reducing cytochrome c than was the complete enzyme. The results suggest that the amino-terminal hydrophobic regions of P450c and the P450/reductase fused enzyme play an important role in their stability and function in the yeast microsomes. By contrast, the amino-terminal-containing P450 reductase appears to be unstable in yeast cells. Altering the size of the hinge regions does not affect the activity of the P450/reductase fused enzyme significantly, but some amino acid changes in this region increase the stability of the fused enzyme slightly.
Yabusaki
Y
Y
Biotechnology Laboratory, Takarazuka Research Center, Sumitomo Chemical Co., Ltd., Hyogo, Japan.
Murakami
H
H
Sakaki
T
T
Shibata
M
M
Ohkawa
H
H
D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D005818:Genetic Engineering; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000502:physiology*; D011994:Recombinant Proteins / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013250:Steroid Hydroxylases / Q000378:metabolism; D013347:Subcellular Fractions / Q000201:enzymology; D014170:Transformation, Genetic
D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D009251:NADPH-Ferrihemoprotein Reductase