Arabidopsis thaliana NAPHP thioredoxin reductase. cDNA characterization and expression of the recombinant protein in Escherichia coli.
Using a clone characterized in the course of a random sequencing programme of Arabidopsis thaliana, two cDNAs encoding plant type cytosolic NADPH-dependent thioredoxin reductase (NTR) have been isolated. Their sequence homology with Escherichia coli NRT (the only thioredoxin reductase of known primary structure) is about 45%. In addition, analysis of the sequence of the encoded polypeptide (333 amino acids) reveals that several motifs are conserved in the FAD, central and NADPH binding domains, suggesting a similar folding of the protein. Definitive proof that the clone ATTHIREDB indeed encodes NTR was obtained by expressing the recombinant protein in E. coli cells. It was observed that plant type NTR was strongly overproduced (about 10 mg homogeneous protein could be purified per liter of culture). The recombinant enzyme is homodimeric, each subunit containing an FAD prosthetic group. Recombinant plant type NTR is as effective as E. coli NTR in the DTNB (5,5'-dithiobis nitrobenzoic acid) reduction reaction, but its affinity for thioredoxin substrates was strikingly different. These results are discussed in relation to the primary structures of NADPH thioredoxin reductases.
Jacquot
J P
JP
Laboratoire de Physiologie Végétale Moléculaire, URA CNRS 1128, Université de Paris-Sud, Orsay, France.
Rivera-Madrid
R
R
Marinho
P
P
Kollarova
M
M
Le Maréchal
P
P
Miginiac-Maslow
M
M
Meyer
Y
Y
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology*; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D010802:Phylogeny; D011994:Recombinant Proteins / Q000096:biosynthesis; D017386:Sequence Homology, Amino Acid; D013880:Thioredoxin-Disulfide Reductase / Q000096:biosynthesis / Q000145:classification / Q000235:genetics*
D011994:Recombinant Proteins; D009249:NADP; D013880:Thioredoxin-Disulfide Reductase