Expression of a glutathione reductase from Brassica rapa subsp. pekinensis enhanced cellular redox homeostasis by modulating antioxidant proteins in Escherichia coli.
Glutathione reductase (GR) is an enzyme that recycles a key cellular antioxidant molecule glutathione (GSH) from its oxidized form (GSSG) thus maintaining cellular redox homeostasis. A recombinant plasmid to overexpress a GR of Brassica rapa subsp. pekinensis (BrGR) in E. coli BL21 (DE3) was constructed using an expression vector pKM260. Expression of the introduced gene was confirmed by semiquantitative RT-PCR, immunoblotting and enzyme assays. Purification of the BrGR protein was performed by IMAC method and indicated that the BrGR was a dimmer. The BrGR required NADPH as a cofactor and specific activity was approximately 458 U. The BrGR-expressing E. coli cells showed increased GR activity and tolerance to H(2)O(2), menadione, and heavy metal (CdCl(2), ZnCl(2) and AlCl(2))-mediated growth inhibition. The ectopic expression of BrGR provoked the co-regulation of a variety of antioxidant enzymes including catalase, superoxide dismutase, glutathione peroxidase, and glucose-6-phosphate dehydrogenase. Consequently, the transformed cells showed decreased hydroperoxide levels when exposed to stressful conditions. A proteomic analysis demonstrated the higher level of induction of proteins involved in glycolysis, detoxification/oxidative stress response, protein folding, transport/binding proteins, cell envelope/porins, and protein translation and modification when exposed to H(2)O(2) stress. Taken together, these results indicate that the plant GR protein is functional in a cooperative way in the E. coli system to protect cells against oxidative stress.
Kim
Il-Sup
IS
Department of Biology, Kyungpook National University, Daegu 702-701, Korea.
Shin
Sun-Young
SY
Kim
Young-Saeng
YS
Kim
Hyun-Young
HY
Yoon
Ho-Sung
HS
D000222:Adaptation, Physiological / Q000187:drug effects; D000975:Antioxidants / Q000378:metabolism*; D029741:Brassica rapa / Q000201:enzymology*; D002104:Cadmium / Q000633:toxicity; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D005980:Glutathione Reductase / Q000302:isolation & purification / Q000378:metabolism*; D006706:Homeostasis / Q000187:drug effects; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000494:pharmacology; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D010084:Oxidation-Reduction / Q000187:drug effects; D018384:Oxidative Stress / Q000187:drug effects; D010957:Plasmids / Q000235:genetics; D020543:Proteome / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013312:Stress, Physiological / Q000187:drug effects; D014170:Transformation, Genetic / Q000187:drug effects; D024483:Vitamin K 3 / Q000494:pharmacology; D015032:Zinc / Q000633:toxicity
D000975:Antioxidants; D020543:Proteome; D011994:Recombinant Proteins; D002104:Cadmium; D024483:Vitamin K 3; D006861:Hydrogen Peroxide; D005980:Glutathione Reductase; D015032:Zinc