Molecules and Cells

Korean Society for Molecular and Cellular Biology (한국분자세포생물학회)
권호 표지
DOI QR코드

DOI QR Code

Expression of a Glutathione Reductase from Brassica rapa subsp. pekinensis Enhanced Cellular Redox Homeostasis by Modulating Antioxidant Proteins in Escherichia coli

  • Received : 2009.09.26
  • Accepted : 2009.10.28
  • Published : 2009.11.30
⟨ Previous Next ⟩

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 semi-quantitative 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 $H_2O_2$, 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 $H_2O_2$ 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.

Keywords

Acknowledgement

Supported by : Rural Development Administration

References

  1. 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 https://doi.org/10.1128/JB.188.9.3371-3381.2006
  2. Benov, L., and Al-Ibraheem, J. (2002). Disrupting Escherichia coli: a comparison of methods. J. Biochem. Mol. Biol. 35, 428-431 https://doi.org/10.5483/BMBRep.2002.35.4.428
  3. 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 https://doi.org/10.1007/s002849900420
  4. 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 https://doi.org/10.1093/nar/20.12.3127
  5. Carmel-Harel, O., and Storz, G. (2000). Roles of the glutathioneand thioredoxin-dependent reduction systems in the Escherichia coli and Saccharomyces cerevisiae responses to oxidative stress. Annu. Rev. Microbiol. 54, 439-461 https://doi.org/10.1146/annurev.micro.54.1.439
  6. 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 https://doi.org/10.1016/j.bbagen.2006.10.013
  7. 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 https://doi.org/10.1128/jb.172.10.5686-5689.1990
  8. 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 https://doi.org/10.1128/jb.175.4.1026-1031.1993
  9. Collinson, L.P., and Dawes, I.W. (1995). Isolation, characterization and overexpression of the yeast gene, GLR1, encoding glutathione reductase. Gene 156, 123-127 https://doi.org/10.1016/0378-1119(95)00026-3
  10. 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
  11. 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 https://doi.org/10.1007/s00299-009-0702-6
  12. 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 https://doi.org/10.1021/bi00357a069
  13. 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 https://doi.org/10.1021/pr700631c
  14. 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 https://doi.org/10.1021/jf071014s
  15. 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 https://doi.org/10.1016/0003-2697(92)90122-N
  16. 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 https://doi.org/10.1074/jbc.270.39.22882
  17. 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
  18. Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685 https://doi.org/10.1038/227680a0
  19. 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 https://doi.org/10.1016/S0167-4781(97)00198-X
  20. 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
  21. 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 https://doi.org/10.1016/j.pep.2005.03.027
  22. 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 https://doi.org/10.1016/j.jinorgbio.2004.04.011
  23. 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 https://doi.org/10.1016/j.femsre.2004.09.004
  24. 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 https://doi.org/10.1016/j.bbrc.2009.02.100
  25. 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 https://doi.org/10.1096/fasebj.13.13.1733
  26. 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 https://doi.org/10.1016/0378-1119(89)90373-9
  27. 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 https://doi.org/10.1128/JB.187.5.1763-1772.2005
  28. 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 https://doi.org/10.1165/ajrcmb.20.2.3367
  29. 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 https://doi.org/10.1111/j.1365-2958.1991.tb01837.x
  30. 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 https://doi.org/10.1111/j.1399-3054.2000.1100405.x
  31. 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 https://doi.org/10.1128/JB.183.24.7173-7181.2001
  32. 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, $H_2O_2$) modulate expression of glutathione reductase (GR) gene from the intertidal copepod Tigriopus japonicus. Aquatic Toxiol. 80, 281-289 https://doi.org/10.1016/j.aquatox.2006.09.005
  33. 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 https://doi.org/10.1016/S0891-5849(99)00214-2
  34. 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 https://doi.org/10.1007/s004250000304
  35. 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 https://doi.org/10.1042/0264-6021:3520071
  36. 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 https://doi.org/10.1074/jbc.273.5.3027
  37. Wheeler, G.L., and Grant, C.M. (2004). Regulation of redox homeostasis in the yeast Saccharomyces cerevisiae. Physiol. Plant 120, 12-20 https://doi.org/10.1111/j.0031-9317.2004.0193.x
  38. 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 https://doi.org/10.1128/JB.186.1.192-199.2004
  39. 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 https://doi.org/10.1016/j.bbrc.2004.11.095
  40. Yu, J., and Zhou, C.Z. (2007). Crystal structure of glutathione reductase Glr1 from the yeast Saccharomyces cerevisiae. Proteins 68, 972-979 https://doi.org/10.1002/prot.21354

Cited by

  1. Enhancing E. coli Tolerance towards Oxidative Stress via Engineering Its Global Regulator cAMP Receptor Protein (CRP) vol.7, pp.12, 2009, https://doi.org/10.1371/journal.pone.0051179
  2. Heavy Metal Tolerance in Plants: Role of Transcriptomics, Proteomics, Metabolomics, and Ionomics vol.6, pp.None, 2015, https://doi.org/10.3389/fpls.2015.01143
  3. Overexpression of Mycothiol Disulfide Reductase Enhances Corynebacterium glutamicum Robustness by Modulating Cellular Redox Homeostasis and Antioxidant Proteins under Oxidative Stress vol.6, pp.None, 2009, https://doi.org/10.1038/srep29491
  4. Drought-Responsive Mechanisms in Plant Leaves Revealed by Proteomics vol.17, pp.10, 2016, https://doi.org/10.3390/ijms17101706
  5. A thioredoxin-dependent peroxiredoxin Q from Corynebacterium glutamicum plays an important role in defense against oxidative stress vol.13, pp.2, 2018, https://doi.org/10.1371/journal.pone.0192674
  6. Myo ‐inositol‐1‐phosphate synthase (Ino‐1) functions as a protection mechanism in Corynebacterium glutamicum under oxidative stress vol.8, pp.5, 2009, https://doi.org/10.1002/mbo3.721
  7. Characterization of recombinant glutathione reductase from Antarctic yeast Rhodotorula mucilaginosa vol.42, pp.12, 2009, https://doi.org/10.1007/s00300-019-02603-3