Abstract
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 H2O2, menadione, and heavy metal (CdCl2, ZnCl2 and AlCl2)-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 H2O2 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.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
Ackerley, D.F., Barak, Y., Lynch, S.V., Curtin, J., and Matin, A. (2006). Effect of chromate stress on Escherichia coli K-12. J. Bacteriol. 188, 3371–3381.
Benov, L., and Al-Ibraheem, J. (2002). Disrupting Escherichia coli: a comparison of methods. J. Biochem. Mol. Biol. 35, 428–431.
Bernstein, C., Bernstein, H., Payne, C.M., Beard, S.E., and Schneider, J. (1999). Bile salt activation of stress response promoters in Escherichia coli. Curr. Microbiol. 39, 68–72.
Bucheler, U.S., Werner, D., and Schirmer, R.H. (1992). Generating compatible translation initiation regions for heterologous gene expression in Escherichia coli by exhaustive periShine-Dalgarno mutagenesis. Human glutathione reductase cDNA as a model. Nucleic Acids Res. 20, 3127–3133.
Carmel-Harel, O., and Storz, G. (2000). Roles of the glutathione- and thioredoxin-dependent reduction systems in the Escherichia coli and Saccharomyces cerevisiae responses to oxidative stress. Annu. Rev. Microbiol. 54, 439–461.
Castro, F.A., Herdeiro, R.S., Panek, A.D., Eleutherio, E.C., and Pereira, M.D. (2007). Menadione stress in Saccharomyces cerevisiae strains deficient in the glutathione transferases. Biochim. Biophys. Acta 1770, 213–220.
Chen, J., Brevet, A., Fromant, M., Leveque, F., Schmitter, J.M., Blanquet, S., and Plateau, P. (1990). Pyrophosphatase is essential for growth of Escherichia coli. J. Bacteriol. 172, 5686–5689.
Chou, J.H., Greenberg, J.T., and Demple, B. (1993). Posttranscriptional repression of Escherichia coli OmpF protein in response to redox stress: positive control of the micF antisense RNA by the soxRS locus. J. Bacteriol. 175, 1026–1031.
Collinson, L.P., and Dawes, I.W. (1995). Isolation, characterization and overexpression of the yeast gene, GLR1, encoding glutathione reductase. Gene 156, 123–127.
Creissen, G.P., and Mullineaux, P.M. (1995). Cloning and characterisation of glutathione reductase cDNAs and identification of two genes encoding the tobacco enzyme. Planta 197, 422–425.
Fan, W., Zhang, Z., and Zhang, Y. (2009). Cloning and molecular characterization of fructose-1,6-bisphosphate aldolase gene regulated by high-salinity and drought in Sesuvium portulacastrum. Plant Cell Rep. 28, 975–984.
Greer, S., and Perham, R.N. (1986). Glutathione reductase from Escherichia coli: cloning and sequence analysis of the gene and relationship to other flavoprotein disulfide oxidoreductases. Biochemistry 25, 2736–2742.
Han, K.Y., Park, J.S., Seo, H.S., Ahn, K.Y., and Lee, J. (2008). Multiple stressor-induced proteome responses of Escherichia coli BL21(DE3). J. Proteome Res. 7, 1891–1903.
Huang, Y.J., Tsai, T.Y., and Pan, T.M. (2007). Physiological response and protein expression under acid stress of Escherichia coli O157:H7 TWC01 isolated from Taiwan. J. Agric. Food Chem. 55, 7182–7191.
Jiang, Z.Y., Hunt, J.V., and Wolff, S.P. (1992). Ferrous ion oxidation in the presence of xylenol orange for detection of lipid hydroperoxide in low density lipoprotein. Anal. Biochem. 202, 384–389.
Jiang, F., Hellman, U., Sroga, G.E., Bergman, B., and Mannervik, B. (1995). Cloning, sequencing, and regulation of the glutathione reductase gene from the cyanobacterium Anabaena PCC 7120. J. Biol. Chem. 270, 22882–22889.
Kubo, A., Sano, T., Saji, H., Tanaka, K., Kondo, N., and Tanaka, K. (1993). Primary structure and properties of glutathione reductase from Arabidopsis thaliana. Plant Cell Physiol. 34, 1259–1266.
Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.
Lee, H., Jo, J., and Son, D. (1998). Molecular cloning and characterization of the gene encoding glutathione reductase in Brassica campestris. Biochim. Biophys. Acta 1395, 309–314.
Lee, H., Won, S.H., Lee, B.H., Park, H.D., Chung, W.I., and Jo, J. (2002). Genomic cloning and characterization of glutathione reductase gene from Brassica campestris var. pekinensis. Mol. Cells 13, 245–251.
Li, M., Huang, W., Yang, Q., Liu, X., and Wu, Q. (2005). Expression and oxidative stress tolerance studies of glutaredoxin from cyanobacterium Synechocystis sp. PCC 6803 in Escherichia coli. Protein Expr. Purif. 42, 85–91.
Martelli, A., and Moulis, J.M. (2004). Zinc and cadmium specifically interfere with RNA-binding activity of human iron regulatory protein 1. J. Inorg. Biochem. 98, 1413–1420.
Mendoza-Cozatl, D., Loza-Tavera, H., Hernandez-Navarro, A., and Moreno-Sanchez, R. (2005). Sulfur assimilation and glutathione metabolism under cadmium stress in yeast, protists and plants. FEMS Microbiol. Rev. 29, 653–671.
Mishra, Y., Chaurasia, N., and Rai, L.C. (2009). AhpC (alkyl hydroperoxide reductase) from Anabaena sp. PCC 7120 protects Escherichia coli from multiple abiotic stresses. Biochem. Biophys. Res. Commun. 381, 606–611.
Mockett, R.J., Sohal, R.S., and Orr, W.C. (1999). Overexpression of glutathione reductase extends survival in transgenic Drosophila melanogaster under hyperoxia but not normoxia. FASEB J. 13, 1733–1742.
Nellemann, L.J., Holm, F., Atlung, T., and Hansen, F.G. (1989). Cloning and characterization of the Escherichia coli phosphoglycerate kinase (pgk) gene. Gene 77, 185–191.
Nishino, K., Honda, T., and Yamaguchi, A. (2005). Genome-wide analyses of Escherichia coli gene expression responsive to the BaeSR two-component regulatory system. J. Bacteriol. 187, 1763–1772.
O’Donovan, D.J., Katkin, J.P., Tamura, T., Husser, R., Xu, X., Smith, C.V., and Welty, S.E. (1999). Gene transfer of mitochondrially targeted glutathione reductase protects H441 cells from t-butyl hydroperoxide-induced oxidant stresses. Am. J. Respir. Cell Mol. Biol. 20, 256–263.
Perry, A.C., Ni Bhriain, N., Brown, N.L., and Rouch, D.A. (1991). Molecular characterization of the gor gene encoding glutathione reductase from Pseudomonas aeruginosa: determinants of substrate specificity among pyridine nucleotide-disulphide oxidoreductases. Mol. Microbiol. 5, 163–171.
Pilon-Smits, E.A., Zhu, Y.L., Sears, T., and Terry, N. (2000). Overexpression of glutathion reductase in Brassica juncea: effects on cadmium accumulation and tolerance. Physiol. Plant 110, 455–460.
Seaver, L.C., and Imlay, J.A. (2001). Alkyl hydroperoxide reductase is the primary scavenger of endogenous hydrogen peroxide in Escherichia coli. J. Bacteriol. 183, 7173–7181.
Seo, J.S., Lee, K.W., Rhee, J.S., Hwang, D.S., Lee, Y.M., Park, H.G., and Park, J.S. (2006). Environmental stressors (salinity, heavy metals, H2O2) modulate expression of glutathione reductase (GR) gene from the intertidal copepod Tigriopus japonicus. Aquatic Toxiol. 80, 281–289.
Spickett, C.M., Smirnoff, N., and Pitt, A.R. (2000). The biosynthesis of erythroascorbate in Saccharomyces cerevisiae and its role as an antioxidant. Free Radic Biol Med 28, 183–192.
Stevens, R.G., Creissen, G.P., and Mullineaux, P.M. (2000). Characterisation of pea cytosolic glutathione reductase expressed in transgenic tobacco. Planta 211, 537–545.
Sugiyama, K., Kawamura, A., Izawa, S., and Inoue, Y. (2000). Role of glutathione in heat-shock-induced cell death of Saccharomyces cerevisiae. Biochem. J. 352, 71–78.
Tamarit, J., Cabiscol, E., and Ros, J. (1998). Identification of the major oxidatively damaged proteins in Escherichia coli cells exposed to oxidative stress. J. Biol. Chem. 273, 3027–3032.
Wheeler, G.L., and Grant, C.M. (2004). Regulation of redox homeostasis in the yeast Saccharomyces cerevisiae. Physiol. Plant 120, 12–20.
Yohannes, E., Barnhart, D.M., and Slonczewski, J.L. (2004). pHdependent catabolic protein expression during anaerobic growth of Escherichia coli K-12. J. Bacteriol. 186, 192–199.
Yoon, H.S., Lee, I.A., Lee, H., Lee, B.H., and Jo, J. (2005). Overexpression of a eukaryotic glutathione reductase gene from Brassica campestris improved resistance to oxidative stress in Escherichia coli. Biochem. Biophys. Res. Commun. 326, 618–623.
Yu, J., and Zhou, C.Z. (2007). Crystal structure of glutathione reductase Glr1 from the yeast Saccharomyces cerevisiae. Proteins 68, 972–979.
Author information
Authors and Affiliations
Corresponding author
About this article
Cite this article
Kim, IS., Shin, SY., Kim, YS. et al. Expression of a glutathione reductase from Brassica rapa subsp. pekinensis enhanced cellular redox homeostasis by modulating antioxidant proteins in Escherichia coli . Mol Cells 28, 479–487 (2009). https://doi.org/10.1007/s10059-009-0168-y
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10059-009-0168-y