Molecular cloning and expression in Saccharomyces cerevisiae of tobacco NADPH-cytochrome P450 oxidoreductase cDNA.
We obtained information on the full length tobacco NADPH-cytochrome P450 oxidoreductase (P450 reductase) by a combination of the cDNA clone pCTR1 and the genomic DNA clone pGTR1. The deduced primary structure consisting of 713 amino acid residues contained sequences corresponding to FMN, FAD, and NADPH-binding regions. Based on this information, we prepared the full-length cDNA pFTR of tobacco P450 reductase by RT-PCR and expressed it in the yeast Saccharomyces cerevisiae. The transformed yeast cells carrying pFTR produced the corresponding mRNA and protein, and had increased cytochrome c reductase activity in the microsomes. An in vitro reconstitution system of the yeast microsomal fractions expressed tobacco P450 reductase and rat P450 1A1 showed an increased 7-ethoxycoumarin O-deethylase activity. These results indicated that tobacco P450 reductase expressed in the yeast microsomes coupled with rat P450 1A1 resulting in an increased monooxygenase activity.
Yamada
T
T
Department of Biological and Environmental Science, Faculty of Agriculture, Kobe University, Japan.
Imaishi
H
H
Oka
A
A
Ohkawa
H
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000302:isolation & purification*; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D008969:Molecular Sequence Data; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics*; D010947:Plants, Toxic; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D014026:Nicotiana / Q000201:enzymology*
D018076:DNA, Complementary; D009251:NADPH-Ferrihemoprotein Reductase