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.
Department of Biological and Environmental Science, Faculty of Agriculture, Kobe University, Japan.
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T
Yamada
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Imaishi
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Oka
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Ohkawa
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