Cloning and expression in Escherichia coli and Saccharomyces cerevisiae of a novel tobacco cytochrome P-450-like cDNA.
A cDNA library constructed from poly(A)+ RNA of tobacco BY2 cells treated with 2,4-dichlorophenoxyacetic acid was screened by using a synthetic oligonucleotide corresponding to the heme binding region of avocado CYP71A1. A cloned 2-kb cDNA designated as cTBP contained an open reading frame of 1593 bp encoding a protein of molecular size of 58916. The deduced amino acid sequence included a cysteine residue corresponding to fifth ligand of heme-Fe at 497th. The coding sequence was expressed under the control of tac promoter and rrnB terminator in Escherichia coli to yield 7 to 10 nmol P450 equivalent per litre of the culture in the presence of delta-aminolevulinic acid. The modified coding sequences in which NH2-terminal residues 2-25 were replaced by the NH2-terminal 18 amino acid residues of microsomal bovine CYP17 were also expressed under the control of ADH promoter and terminator in Saccharomyces cerevisiae to yield 29 and 30 pmol of P450 equivalent/mg protein in the microsomal fraction, respectively. On co-expression of each of the modified coding sequences and yeast NADPH-cytochrome P-450 oxidoreductase gene, the yeast microsomes exhibited 7-ethoxycoumarin O-deethylase activity. Based on these results, tobacco cTBP was found to encode a novel P450-like species with a monooxygenese activity related to xenobiotic metabolism.
Sugiura
M
M
Department of Biological and Environmental Science, Kobe University, Japan.
Sakaki
T
T
Yabusaki
Y
Y
Ohkawa
H
H
D015084:2,4-Dichlorophenoxyacetic Acid / Q000494:pharmacology; D015660:7-Alkoxycoumarin O-Dealkylase / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics*; D015342:DNA Probes; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D010947:Plants, Toxic; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013053:Spectrophotometry; D014026:Nicotiana / Q000201:enzymology / Q000235:genetics*
D015342:DNA Probes; D018076:DNA, Complementary; D011994:Recombinant Proteins; D015084:2,4-Dichlorophenoxyacetic Acid; D003577:Cytochrome P-450 Enzyme System; D015660:7-Alkoxycoumarin O-Dealkylase