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Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
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Purification and Physicochemical Characterization of a Recombinant Phospholipid Hydroperoxide Glutathione Peroxidase from Oryza sativa

  • Wang, Zebin (Laboratory of Molecular Biology and Protein Science Laboratory of the Ministry of Education, Department of Biological Sciences and Biotechnology, Tsinghua University) ;
  • Wang, Feng (Laboratory of Molecular Biology and Protein Science Laboratory of the Ministry of Education, Department of Biological Sciences and Biotechnology, Tsinghua University) ;
  • Duan, Rui (Laboratory of Molecular Biology and Protein Science Laboratory of the Ministry of Education, Department of Biological Sciences and Biotechnology, Tsinghua University) ;
  • Liu, Jin-Yuan (Laboratory of Molecular Biology and Protein Science Laboratory of the Ministry of Education, Department of Biological Sciences and Biotechnology, Tsinghua University)
  • Published : 2007.05.31
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Abstract

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is an unique antioxidant enzyme that directly reduces lipid hydroperoxides in biomembranes. In the present work, the entire encoding region for Oryza sativa PHGPx was expressed in Escherichia coli M15, and the purified fusion protein showed a single band with 21.0 kD and pI = 8.5 on SDS- and IFE-PAGE, respectively. Judging from CD and fluorescence spectroscopy, this protein is considered to have a well-ordered structure with 12.2% $\alpha$-helix, 30.7%$\beta$-sheet, 18.5% $\delta$-turn, and 38.5% random coil. The optimum pH and temperature of the enzyme activity were pH 9.3 and 27$^{\circ}C$. The enzyme exhibited the highest affinity and catalytical efficiency to phospholipid hydroperoxide employing GSH or Trx as electron donor. Moreover, the protein displayed higher GSH-dependent activity towards t-Butyl-OOH and $H_2O_2$. These results show that OsPHGPx is an enzyme with broad specificity for hydroperoxide substrates and yielded significant insight into the physicochemical properties and the dynamics of OsPHGPx.

Keywords

References

  1. Agrawal, G. K., Rakwal, R., Jwa, N.-S. and Agrawal, V. P. (2002) Effects of signaling molecules, protein phosphatase inhibitors and blast pathogen (Magnaporthe grisea) on the mRNA level of a rice (Oryza sativa L.) phospholipid hydroperoxide glutathione peroxidase (OsPHGPx) gene in seedling leaves. Gene 283, 227-236. https://doi.org/10.1016/S0378-1119(01)00854-X
  2. Bao, Y. P., Chambers, S. J. and Williamson, G. (1995) Direct separation of hydroperoxy- and hydrooxy-phosphtidylcholine derivatives: application to the assay of phospholipid hydroperoxide glutathione peroxidase. Anal. Biochem. 224, 395-399. https://doi.org/10.1006/abio.1995.1056
  3. Beeor-Tzahar, T., Ben-Hayyim, G., Holland, D., Faltin, Z. and Eshdat, Y. (1995) A stress-associated citrus protein is a distinct plant phospholipid hydroperoxide glutathione peroxidase. FEBS Lett. 366, 151-155. https://doi.org/10.1016/0014-5793(95)00521-A
  4. Chen, S., Vaghchhipawala, Z., Li, W., Asard, H. and Dickman, M. B. (2004) Tomato phospholipid hydroperoxide glutathione peroxidase inhibits cell death induced by bax and oxidative stresses in yeast and plants. Plant Physiol. 135, 1630-1641. https://doi.org/10.1104/pp.103.038091
  5. Churin, Y., Schilling, S. and Borner, T. (1999) A gene family encoding glutathione peroxidase homologues in Hordeum vulgare (barley). FEBS Lett. 459, 33-38. https://doi.org/10.1016/S0014-5793(99)01208-9
  6. Eshdat, Y., Holland, D., Faltin, Z. and Ben-Hayyim, G. (1997) Plant glutathione peroxidases. Physiol. Plant 100, 234-240. https://doi.org/10.1111/j.1399-3054.1997.tb04779.x
  7. Flohe, L. and Gunzler, W. A. (1984) Assays of glutathione peroxidase. Methods Enzymol. 105, 114-121. https://doi.org/10.1016/S0076-6879(84)05015-1
  8. Hazebrouck, S., Camoin, L., Faltin, Z., Strosberg, A. D. and Eshdat, Y. (2000) Substituting selenocysteine for catalytic cysteine 41 enhances enzymatic activity of plant phospholipid hydroperoxide glutathione peroxidase expressed in E. coli. J. Biol. Chem. 275, 28715-28721. https://doi.org/10.1074/jbc.M004985200
  9. Herbette, S., Lenne, C., Leblanc, N., Julien, J. L., Drevet, J. R. and Roeckel-Drevet, P. (2002) Two GPX-like proteins from Lycopersicon esculentum and Helianthus annuus are antioxidant enzymes with phospholipid hydroperoxide glutathione peroxidase and thioredoxin peroxidase activities. Eur. J. Biochem. 269, 2414-2420. https://doi.org/10.1046/j.1432-1033.2002.02905.x
  10. Holland, D., Ben-Hayyim, G., Faltin, Z., Camoin, L., Strosberg, A. D. and Eshdat, Y. (1993) Molecular characterization of salt stress-associated protein in citrus: protein and cDNA sequence homology to mammalian glutathione peroxidases. Plant Mol. Biol. 21, 923-927. https://doi.org/10.1007/BF00027124
  11. Imai, H. and Nakagawa, Y. (2003) Biological significance of phospholipid hydroperoxide glutathione peroxidase (PHGPx, Gpx4) in mammalian cells. Free Radic. Bio. Med. 34, 145-169. https://doi.org/10.1016/S0891-5849(02)01197-8
  12. Kvaratskhelia, M., Winkel, C. and Thorneley, R. N. F. (1997) Purification and characterization of a novel class III peroxidase isoenzyme from tea leaves. Plant Physiol. 114, 1237-1245. https://doi.org/10.1104/pp.114.4.1237
  13. Laemmli, U. K. (1970) Cleavages of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. https://doi.org/10.1038/227680a0
  14. Li, W.-J., Feng, H., Fan, J.-H., Zhang, R.-Q., Zhao, N.-M. and Liu, J.-Y. (2000) Molecular cloning and expression of a phospholipid hydroperoxide glutathione peroxidase homolog in Oryza sativa. Biochim. Biophys. Acta 1493, 225-230. https://doi.org/10.1016/S0167-4781(00)00152-4
  15. Liu, J. Y., Le, Y., Ye, B., Zhen, Y., Zhu, C., Shen, J. and Zhang, R. (2002) Tabtoxin-resistant protein: overexpression, purification, and characterization. Protein Expr. Purif. 24, 439-444. https://doi.org/10.1006/prep.2001.1586
  16. Kim, J. A., Park, S., Kim, K., Rhee, S. G. and Kang, S. W. (2005) Activity assay of mammalian 2-cys peroxiredoxins using yeast thioredoxin reductase system. Anal. Biochem. 338, 216-223. https://doi.org/10.1016/j.ab.2004.12.008
  17. Maiorino, M., Gregolin, C. and Ursini, F. (1990) Phospholipid hydroperoxide glutathione peroxidase. Methods Enzymol. 186, 448-457. https://doi.org/10.1016/0076-6879(90)86139-M
  18. Mullineaux, P. M., Karpinski, S., Jimenez, A., Cleary, S. P., Robinson, C. and Creissen, G. P. (1998) Identification of cDNAs encoding plastid-targeted glutathione peroxidase. Plant J. 13, 375-379. https://doi.org/10.1046/j.1365-313X.1998.00052.x
  19. Sreenivasulu, N., Miranda, M., Prakash, H. S., Wobus, U. and Weschke, W. (2004) Transcriptome changes in foxtail millet genotypes at high salinity: identification and characterization of a PHGPX gene specifically upregulated by NaCl in a salttolerant line. J. Plant Physiol. 161, 467-477. https://doi.org/10.1078/0176-1617-01112
  20. Sugimoto, M., Furui, S. and Suzuki, Y. (1997) Molecular cloning and characterization of a cDNA encoding putative phospholipid hydroperoxide glutathione peroxidase from spinach. Biosci. Biotechnol. Biochem. 61, 1379-1381. https://doi.org/10.1271/bbb.61.1379
  21. Sugimoto, M. and Sakamoto, W. (1997) Putative phospholipid hydroperoxide glutathione peroxidase gene from Arabidopsis thaliana induced by oxidative stress. Genes Genet. Syst. 72, 311-316. https://doi.org/10.1266/ggs.72.311
  22. Ursini, F., Heim, S., Kiess, M., Maiorino, M., Roveri, A., Wissing, J. and Flohe, L. (1999) Dual function of the selenoprotein PHGPx during sperm maturation. Science 285, 1393-1396. https://doi.org/10.1126/science.285.5432.1393
  23. Ursini, F., Mariorino, M., Brigelius-Flohe, R., Aumann, K. D., Roveri, A., Schomburg, D. and Flohe, L. (1995) Diversity of glutathione peroxidase. Methods Enzymol. 252, 38-53. https://doi.org/10.1016/0076-6879(95)52007-4
  24. Ursini, F., Maiorino, M., Valente, M., Ferri, L. and Gregolin, C. (1982) Purification from pig liver of a protein which protects liposomes and biomembranes from peroxidative degradation and exhibits glutathione peroxidase activity on phosphatidylcholine hydroperoxides. Biochim. Biophys. Acta 710, 197-211. https://doi.org/10.1016/0005-2760(82)90150-3
  25. Yang, J. T., Wu, C-S. C. and Martinez, H. M. (1986) Calculation of protein conformation from circular dichroism. Methods Enzymol. 130, 208-269. https://doi.org/10.1016/0076-6879(86)30013-2
  26. Yang, X.-D., Li, W.-J. and Liu, J.-Y. (2005) Isolation and characterization of a novel PHGPx gene in Raphanus sativus. Biochim. Biophys. Acta 1728, 199-205. https://doi.org/10.1016/j.bbaexp.2005.02.003
  27. Yang, X.-D. and Liu, J.-Y. (2005) Micropreparation of a native PHGPx protein from radish seedlings by immunoaffinity chromatography. Prog. Biochem. Biophysic. 32, 794-799.

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