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Biochemical and Molecular Characterization of a Novel Cu/Zn Superoxide Dismutase from Amaranthus hypochondriacus L.: an Intrinsically Disordered Protein

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Abstract

A novel Cu/ZnSOD from Amaranthus hypochondriacus was cloned, expressed, and characterized. Nucleotide sequence analysis showed an open reading frame (ORF) of 456 bp, which was predicted to encode a 15.6-kDa molecular weight protein with a pI of 5.4. Structural analysis showed highly conserved amino acid residues involved in Cu/Zn binding. Recombinant amaranth superoxide dismutase (rAhSOD) displayed more than 50 % of catalytic activity after incubation at 100 °C for 30 min. In silico analysis of Amaranthus hypochondriacus SOD (AhSOD) amino acid sequence for globularity and disorder suggested that this protein is mainly disordered; this was confirmed by circular dichroism, which showed the lack of secondary structure. Intrinsic fluorescence studies showed that rAhSOD undergoes conformational changes in two steps by the presence of Cu/Zn, which indicates the presence of two binding sites displaying different affinities for metals ions. Our results show that AhSOD could be classified as an intrinsically disordered protein (IDP) that is folded when metals are bound and with high thermal stability.

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Abbreviations

AhSOD:

Amaranthus hypochondriacus SOD

CD:

Circular dichroism

IDP:

Intrinsically disordered protein

SOD:

Superoxide dismutase

ROS:

Reactive oxygen species

ICP-OES:

Inductively coupled plasma-optical emission spectrometry

References

  1. Fridovich, I. (1995). Superoxide radical and superoxide dismutases. Annual Review of Biochemistry, 64, 97–112.

    Article  CAS  Google Scholar 

  2. Droillard, M. J., & Paulin, A. (1990). Isozymes of superoxide dismutase in mitochondria and peroxisomes isolated from petals of carnation (Dianthus caryophyllus) during senescence. Plant Physiology, 94, 1187–1192.

    Article  CAS  Google Scholar 

  3. Kanematsu, S., & Asada, K. (1990). Characteristic amino acid sequences of chloroplast and cytosol isozymes of CuZn-superoxide dismutase in spinach, rice and horsetail. Plant & Cell Physiology, 31, 99–112.

    CAS  Google Scholar 

  4. Corpas, F. J., Sandalio, L. M., del Rio, L., & Trelease, R. N. (1998). Copper-zinc superoxide dismutase is a constituent enzyme of the matrix of peroxisomes in the cotyledons of oilseed plants. New Phytologist, 138, 307–314.

    Article  CAS  Google Scholar 

  5. Alscher, R. G., Erturk, N., & Heath, L. S. (2002). Role of superoxide dismutases (SODs) in controlling oxidative stress in plants. Journal of Experimental Botany, 372, 1331–1341.

    Article  Google Scholar 

  6. Miller, A. F. (2012). Superoxide dismutases: ancient enzymes and new insights. FEBS Letters, 586, 585–595.

    Article  CAS  Google Scholar 

  7. Rao, M. B., Tanksale, A. M., Ghatge, M. S., & Deshpande, V. V. (1998). Molecular and biotechnological aspects of microbial proteases. Microbiology and Molecular Biology Reviews, 62, 597–635.

    CAS  Google Scholar 

  8. Huerta-Ocampo, J. A., León-Galván, M. F., Ortega-Cruz, L. B., Barrera-Pacheco, A., De León-Rodríguez, A., Mendoza-Hernández, G., & Barba de la Rosa, A. P. (2011). Water stress induces up-regulation of DOF1 and MIF1 transcription factors and down-regulation of proteins involved in secondary metabolism in amaranth roots (Amaranthus hypochondriacus L.). Plant Biology, 13, 472–482.

    Article  CAS  Google Scholar 

  9. Kauffman, C. S., & Weber, L. E. (1990). Grain amaranth. In J. Janick, & J. E. Simon (Eds.), Advances in new crops (pp. 127–139). Portland, OR: Timber Press.

    Google Scholar 

  10. Omami, E. N., Hammes, P. S., & Robbertse, P. J. (2006). Differences in salinity tolerance for growth and water-use efficiency in some amaranth (Amaranthus spp.) genotypes. New Zealand Journal of Crop and Horticultural Science, 34, 11–22.

    Article  Google Scholar 

  11. Sharma, S., Bahuguna, S., Kaur, N., & Chaudhary, N. (2014). Biochemical aspects of superoxide dismutase isolated from Amaranthus spinosus: a therapeutically important plant. International Journal of Genetic Engineering and Biotechnology, 5, 35–42.

    Google Scholar 

  12. Bouftira, I., Abdelly, C., & Sfar, S. (2008). Characterization of cosmetic cream with Mesembryanthemum crystallinum plant extract: influence of formulation composition on physical stability and anti-oxidant activity. International Journal of Cosmetic Science, 30, 443–452.

    Article  CAS  Google Scholar 

  13. Johnson, F., & Giulivi, C. (2005). Superoxide dismutases and their impact upon human health. Molecular Aspects of Medicine, 26, 340–352.

    Article  CAS  Google Scholar 

  14. Angelova, M., Dolashka-Angelova, P., Ivanova, E., Serkedjieva, J., Slokoska, L., Pashova, S., Toshkova, R., Vassilev, S., Simeonov, I., Hartmann, H. J., Stoeva, S., Weser, U., & Voelter, W. (2001). A novel glycosylated Cu/Zn-containing superoxide dismutase: production and potential therapeutic effect. Microbiology, 147, 1641–1650.

    CAS  Google Scholar 

  15. Hileman, E. A., Achanta, G., & Huang, P. (2001). Superoxide dismutase: an emerging target for cancer therapeutics. Expert Opinion on Therapeutic Targets, 5, 697–710.

    Article  CAS  Google Scholar 

  16. Galaleldeen, A., Strange, R., Whitson, L. J., Antonuk, S., Narayana, N., Taylor, A. B., Schuermann, J. P., Holloway, S. P., Hasnain, S. S., & Hart, P. J. (2009). Structure and biophysical properties of metal-free pathogenic SOD1 mutants A4V and G93A. Archives of Biochemistry and Biophysics, 492, 40–47.

    Article  CAS  Google Scholar 

  17. Roberts, B. R., Tainer, J. A., Getzoff, E. D., Malencik, D. A., Anderson, S. R., Bomben, V. C., Meyers, K. R., Karplus, P. A., & Beckman, J. S. (2007). Structural characterization of zinc-deficient human superoxide dismutase and implications for ALS. Journal of Molecular Biology, 373, 877–890.

    Article  CAS  Google Scholar 

  18. Muid, K. A., Karakaya, H. C., & Koc, A. (2014). Absence of superoxide dismutase activity causes nuclear DNA fragmentation during the aging process. Biochemical and Biophysical Research Communications, 442, 260–263.

    Article  Google Scholar 

  19. Lods, L. M., Dres, C., Johnson, C., Scholz, D. B., & Brooks, G. J. (2000). International Journal of Cosmetic Science, 22, 85–94.

    Article  CAS  Google Scholar 

  20. Saito, N., & Nei, M. (1987). The neighbor-joining method: a new method for reconstruction phylogenetic trees. Molecular Biology and Evolution, 4, 406–425.

    Google Scholar 

  21. Tamura, K., Stecher, G., Peterson, D., Filipski, A., & Kumar, S. (2013). MEGA6: molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution, 30, 2725–2729.

    Article  CAS  Google Scholar 

  22. Beaucham, C., & Fridovich, I. (1971). Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Analytical Biochemistry, 44, 276–278.

    Article  Google Scholar 

  23. Vyas, D., Kumar, S., & Ahuja, P. S. (2007). Tea (Camellia sinensis) clones with shorter periods of winter dormancy exhibit lower accumulation of reactive oxygen species. Tree Physiology, 27, 1253–1259.

    Article  CAS  Google Scholar 

  24. Sreerama, N., & Woody, R. W. (2000). Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set. Analytical Biochemistry, 287, 252–260.

    Article  CAS  Google Scholar 

  25. Whitmore, L., & Wallace, B. A. (2008). Protein secondary structure analyses from circular dichroism spectroscopy: methods and reference databases. Biopolymers, 89, 392–400.

    Article  CAS  Google Scholar 

  26. Linding, R., Jensen, L. J., Diella, F., Bork, P., Gibson, T. J., & Russell, R. B. (2003). Protein disorder prediction: implications for structural proteomics. Structure, 11, 1453–1459.

    Article  CAS  Google Scholar 

  27. Emsley, B., Lohkamp, B., Scott, W. G., & Cowtan, K. (2010). Features and development of coot. Acta Crystallographica Section D: Biological Crystallography, 66, 486–501.

    Article  CAS  Google Scholar 

  28. Pettersen, E. F., Goddard, T. D., Huang, C. C., Couch, G. S., Greenblart, D. M., Meng, E. C., & Ferrin, T. E. (2004). UCSF chimera—a visualization system for exploratory research and analysis. Journal of Computational Chemistry, 25, 1605–1612.

    Article  CAS  Google Scholar 

  29. Nakamura, R., Nakamura, R., Adachi, R., Hachisuka, A., Yamada, A., Ozeki, Y., & Teshima, R. (2014). Differential analysis of protein expression in RNA-binding-protein transgenic and parental rice seeds cultivated under salt stress. Journal of Proteome Research, 13, 489–495.

    Article  CAS  Google Scholar 

  30. Diaz-Vivancos, P., Faize, M., Barba-Espin, G., Faize, L., Petri, C., Hernández, J. A., & Burgos, L. (2013). Ectopic expression of cytosolic superoxide dismutase and ascorbate peroxidase leads to salt stress tolerance in transgenic plums. Plant Biotechnology Journal, 11, 976–985.

    Article  CAS  Google Scholar 

  31. Fridovich, I. (1986). Superoxide dismutases. Advances in Enzymology, 58, 61–97.

    CAS  Google Scholar 

  32. Sundaram, S., Khanna, S., & Khanna-Chopra, R. (2009). Purification and characterization of thermostable monomeric chloroplastic Cu/Zn superoxide dismutase from Chenopodium murale. Physiology and Molecular Biology of Plants, 15, 199–209.

    Article  CAS  Google Scholar 

  33. Uversky, V. N. (2013). Unusual biophysics of intrinsically disordered proteins. Biochimica et Biophysica Acta, 184, 932–951.

    Article  Google Scholar 

  34. Lin, C. T., Kuo, T. J., Shaw, J. F., & Kao, M. C. (1999). Characterization of the dimer-monomer equilibrium of the papaya copper/zinc superoxide dismutase and its equilibrium shift by a single amino acid mutation. Journal of Agricultural and Food Chemistry, 47, 2944–2949.

    Article  CAS  Google Scholar 

  35. Lin, C. T., Lin, M. T., Chen, Y. T., & Shaw, J. F. (1995). Subunit interaction enhances enzyme activity and stability of sweet potato cytosolic Cu/Zn-superoxide dismutase purified by a His-tagged recombinant protein method. Plant Molecular Biology, 28, 303–311.35.

    Article  CAS  Google Scholar 

  36. Zhang, L. Q., Guo, F. X., Xian, H. Q., Wang, X. J., Li, A. N., & Li, D. C. (2011). Expression of a novel thermostable Cu, Zn-superoxide dismutase from Chaetomium thermophilum in Pichia pastoris and its antioxidant properties. Biotechnology Letters, 33, 1127–1132.

    Article  CAS  Google Scholar 

  37. Zhu, Y., Wang, G., Ni, H., Xiao, A., & Cai, H. (2014). Cloning and characterization of a new manganese superoxide dismutase from deep-sea thermophile Geobacillus sp. EPT3. World Journal of Microbiology and Biotechnology, 30, 1347–135737.

    Article  CAS  Google Scholar 

  38. Khanna-Chopra, R., & Sabarinath, R. (2004). Heat-stable chloroplastic Cu/Zn superoxide dismutase in Chenopodium murale. Biochemical and Biophysical Research Communications, 320, 1187–1192.

    Article  CAS  Google Scholar 

  39. Yogavel, M., Mishra, P. C., Gill, J., Bhardwaj, P. K., Dutt, S., Kumar, S., Ahuja, P. S., & Sharma, A. (2008). Structure of a superoxide dismutase and implications for copper-ion chelation. Acta Crystallographica. Section D: Biological Crystallography, 64, 892–901.

    Article  CAS  Google Scholar 

  40. Kumar, A., Dutt, S., Bagler, G., Singh, P., Ahuja, S., & Kumar, S. (2012). Engineering a thermo-stable superoxide dismutase functional at sub-zero to >50 °C, which also tolerates autoclaving. Science Reports, 2, 387 doi:10-1038/srep00387.

    Google Scholar 

  41. Rosa, C. E., Bianchini, A., & Monserrat, J. M. (2008). Antioxidant responses of Laeonereis acuta (Polychaeta) after exposure to hydrogen peroxide. Brazilian Journal of Medical and Biological Research, 41, 117–121.

    Article  Google Scholar 

  42. Zhang, S., Li, X. R., Xu, H., Cao, Y., Ma, S. H., Cao, Y., & Qiao, D. (2014). Molecular cloning and functional characterization of MnSOD from Dunaliella salina. Journal of Basic Microbiology, 54, 438–447.

    Article  CAS  Google Scholar 

  43. Gomez, J. M., Jiménez, A., Olmos, E., & Sevilla, F. (2004). Location and effects of long-term NaCl stress on superoxide dismutase and ascorbate peroxidase isoenzymes of pea (Pisum sativum cv Puget) chloroplasts. Journal of Experimental Botany, 55, 119–130.

    Article  CAS  Google Scholar 

  44. Park, C., & Marqusee, S. (2004). Probing the high energy states in proteins by proteolysis. Journal of Molecular Biology, 343, 1467–1476.

    Article  CAS  Google Scholar 

  45. Daniel, R. M., Cowan, D. A., Morgan, H. W., & Curran, M. P. A. (1982). Correlation between protein thermostability and resistance to proteolysis. Biochemical Journal, 207, 641–644.

    Article  CAS  Google Scholar 

  46. Parcell, D. A., & Sauer, R. T. (1989). The structural stability of a protein is an important determinant of its proteolytic susceptibility in Escherichia coli. The Journal of Biological Chemistry, 264, 7590–7595.

    Google Scholar 

  47. Li, R. H., Liu, G. B., Wang, H., & Zheng, Y. Z. (2013). Effects of Fe3+ and Zn2+ on the structural and thermodynamic properties of a soybean SR protein. Bioscience, Biotechnology, and Biochemistry, 77, 475–481.

    Article  CAS  Google Scholar 

  48. Hara, M., Shinoda, Y., Tanaka, Y., & Kuboi, T. (2009). DNA binding of citrus dehydrin promoted by zinc ion. Plant, Cell & Environment, 32, 532–554.

    Article  CAS  Google Scholar 

  49. Choi, I., Song, H. D., Lee, S., Yang, Y. I., Nam, J. H., Kim, S. J., Sung, J. J., Kang, T. K., & Yi, J. (2011). Direct observation of defects and increased ion permeability of a membrane induced by structurally disordered Cu/Zn-superoxide dismutase aggregates. PLoS ONE, 6, e28982.

    Article  CAS  Google Scholar 

  50. Schulenburg, C., & Hilvert, D. (2013). Protein conformational disorder and enzyme catalysis. Topics in Current Chemistry, 337, 69–9451.

    Article  Google Scholar 

  51. Sun, X., Rikkerink, E. H. A., Jones, W. T., & Uversky, V. N. (2013). Multifarious roles of intrinsic disorder in proteins illustrates its broad impact on plant biology. The Plant Cell, 25, 38–55.

    Article  CAS  Google Scholar 

  52. Battaglia, M., Olvera-Carrilo, Y., Garciarrubio, A., Campos, F., & Covarrubias, A. A. (2008). The enigmatic LEA proteins and other hydrophilins. Plant Physiology, 148, 6–24.

    Article  CAS  Google Scholar 

  53. Hincha, D. K., & Thalhammer, A. (2012). LEA proteins: IDPs with versatile functions in cellular dehydration tolerance. Biochemical Society Transactions, 40, 1000–1003.

    Article  CAS  Google Scholar 

  54. Kalifa, Y., Gilad, A., Konrad, Z., Zaccai, M., Scolnik, P. A., & Bar-zvi, D. (2004). The water-and salt-stress-regulated Asr1 (abscisic acid stress ripening) gene encodes a zinc-dependent DNA-binding protein. Biochemical Journal, 381, 373–378.

    Article  CAS  Google Scholar 

  55. Tompa, P. (2012). Intrinsically disordered proteins: a 10-year recap. Trends in Biochemical Sciences, 37, 509–516.

    Article  CAS  Google Scholar 

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Acknowledgments

GMM thanks Dr. Samuel Lara for helpful discussions and his continued interest in this work and also CONACYT for her postdoctoral fellowship 0156497. We thank Alberto Barrera-Pacheco for his technical support. JGS thanks CONACYT grant 0156497.

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Authors and Affiliations

  1. División Biología Molecular, IPICyT, Instituto Potosino de Investigación Científica y Tecnológica, Camino a la Presa San José 2055, Col. Lomas 4a. Sección, 78216, San Luis Potosí, SLP, Mexico

    Gabriela M. Montero-Morán, Miguel A. Cervantes-González, José Á. Huerta-Ocampo, Antonio De León-Rodríguez & Ana P. Barba de la Rosa

  2. Instituto de Física, Universidad Autónoma de San Luis Potosí, Manuel Nava 6, zona Universitaria, 78290, San Luis Potosí, SLP, Mexico

    José G. Sampedro

  3. Instituto de Biotecnología, Departamento de Ingeniería Celular y Biocatálisis, UNAM, AP 510-3, 62271, Cuernavaca, Morelos, Mexico

    Gloria Saab-Rincón

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  1. Gabriela M. Montero-Morán
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  2. José G. Sampedro
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  3. Gloria Saab-Rincón
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  5. José Á. Huerta-Ocampo
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  6. Antonio De León-Rodríguez
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  7. Ana P. Barba de la Rosa
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Correspondence to Ana P. Barba de la Rosa.

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Montero-Morán, G.M., Sampedro, J.G., Saab-Rincón, G. et al. Biochemical and Molecular Characterization of a Novel Cu/Zn Superoxide Dismutase from Amaranthus hypochondriacus L.: an Intrinsically Disordered Protein. Appl Biochem Biotechnol 176, 2328–2345 (2015). https://doi.org/10.1007/s12010-015-1721-0

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