Biochemistry (Moscow)

, Volume 75, Issue 3, pp 375–381 | Cite as

Structural stability and functional analysis of L-asparaginase from Pyrococcus furiosus

  • S. Bansal
  • D. Gnaneswari
  • P. Mishra
  • B. KunduEmail author


We report studies on an L-asparaginase from Pyrococcus furiosus, cloned and expressed in Escherichia coli and purified to homogeneity. Protein stability and enzyme kinetic parameters were determined. The enzyme was found to be thermostable, natively dimeric, and glutaminase-free, with optimum activity at pH 9.0. It showed a K m of 12 mM and a substrate inhibition profile above 20 mM L-asparagine. Urea could not induce unfolding and enzyme inactivation; however, with guanidine hydrochloride (GdnCl) a two-state unfolding pattern was observed. Reduced activity and an altered near-UV-CD signal for protein at low GdnCl concentration (1 M) suggested tertiary structural changes at the enzyme active site. A homology three-dimensional model was developed and the structural information was combined with activity and stability data to give functional clues about the asparaginase.

Key words

Pyrococcus furiosus L-asparaginase hyperthermophile chemical denaturation homology model stability enzyme kinetics 




EcAII, ErA, PfA, PhA, TaqA, and TthA

asparaginases from Escherichia coli, Erwinia chrysanthemi, Pyrococcus furiosus, P. horikoshii, Thermus aquaticus, and T. thermophilus


guanidine hydrochloride


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Supplementary material

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  1. 1.
    Mashburn, L. T., and Wriston, J. C., Jr. (1964) Arch. Biochem. Biophys., 105, 450–452.CrossRefPubMedGoogle Scholar
  2. 2.
    Verma, N., Kumar, K., Kaur, G., and Anand, S. (2007) Artif. Cells Blood Substit. Immobil. Biotechnol., 35, 449–456.CrossRefPubMedGoogle Scholar
  3. 3.
    Yun, M. K., Nourse, A., White, S. W., Rock, C. O., and Heath, R. J. (2007) J. Mol. Biol., 369, 794–811.CrossRefPubMedGoogle Scholar
  4. 4.
    Sanches, M., Krauchenco, K., and Polikarpov, I. (2007) Curr. Chem. Biol., 1, 75–86.CrossRefGoogle Scholar
  5. 5.
    Ollenschlager, G., Roth, E., Linkesch, W., Jansen, S., Simmel, A., and Modder, B. (1988) Eur. J. Clin. Invest., 18, 512–516.CrossRefPubMedGoogle Scholar
  6. 6.
    Ashworth, L. A., and MacLennan, A. P. (1974) Cancer Res., 34, 1353–1359.PubMedGoogle Scholar
  7. 7.
    Stecher, A. L., de Deus, P. M., Polikarpov, I., and Abrahao-Neto, J. (1999) Pharm. Acta Helv., 74, 1–9.CrossRefPubMedGoogle Scholar
  8. 8.
    Li, L. Z., Xie, T. H., Li, H. J., Qing, C., Zhang, G. M., and Sun, M. S. (2007) Enzyme Microb. Technol., 41, 523–527.CrossRefGoogle Scholar
  9. 9.
    Lubkowski, J., Palm, G. J., Gilliland, G. L., Derst, C., Rohm, K. H., and Wlodawer, A. (1996) Eur. J. Biochem., 241, 201–207.CrossRefPubMedGoogle Scholar
  10. 10.
    Wriston, J. C., Jr., and Yellin, T. O. (1973) Adv. Enzymol. Relat. Areas Mol. Biol., 39, 185–248.CrossRefPubMedGoogle Scholar
  11. 11.
    Amrein, T. M., Schonbachler, B., Escher, F., and Amado, R. (2004) J. Agric. Food Chem., 52, 4282–4288.CrossRefPubMedGoogle Scholar
  12. 12.
    Rosen, J., and Hellenas, K. E. (2002) Analyst, 127, 880–882.CrossRefPubMedGoogle Scholar
  13. 13.
    Mottram, D. S., Wedzicha, B. L., and Dodson, A. T. (2002) Nature, 419, 448–449.CrossRefPubMedGoogle Scholar
  14. 14.
    Pedreschi, F., Kaack, K., and Granby, K. (2008) Food Chem., 109, 386–392.CrossRefGoogle Scholar
  15. 15.
    Ciesarova, Z., Kiss, E., and Boegl, P. (2006) J. Food Nutr. Res., 42, 141–146.Google Scholar
  16. 16.
    Kuilman, M., and Wilms, L. (2007) Toxicol. Lett., 172, S196–S197.CrossRefGoogle Scholar
  17. 17.
    Morales, F., Capuano, E., and Fogliano, V. (2008) Ann. N. Y. Acad. Sci., 1126, 89–100.CrossRefPubMedGoogle Scholar
  18. 18.
    Ehrman, M., Cedar, H., and Schwartz, J. H. (1971) J. Biol. Chem., 246, 88–94.PubMedGoogle Scholar
  19. 19.
    Dunlop, P. C., Meyer, G. M., Ban, D., and Roon, R. J. (1978) J. Biol. Chem., 253, 1297–1304.PubMedGoogle Scholar
  20. 20.
    Kotzia, G. A., and Labrou, N. E. (2005) J. Biotechnol., 119, 309–323.CrossRefPubMedGoogle Scholar
  21. 21.
    Kotzia, G. A., and Labrou, N. E. (2007) J. Biotechnol., 127, 657–669.CrossRefPubMedGoogle Scholar
  22. 22.
    Tollersrud, O. K., and Aronson, N. N., Jr. (1989) Biochem. J., 260, 101–108.PubMedGoogle Scholar
  23. 23.
    Neerunjun, E. D., and Gregoriadis, G. (1976) Biochem. Soc. Trans., 4, 133–134.PubMedGoogle Scholar
  24. 24.
    Chang, T. M. S. (1984) Appl. Biochem. Biotechnol., 10, 5–24.CrossRefPubMedGoogle Scholar
  25. 25.
    Wileman, T. E., Foster, R. L., and Elliott, P. N. (1986) J. Pharm. Pharmacol., 38, 264–271.PubMedGoogle Scholar
  26. 26.
    Poznansky, M. J., Shandling, M., Salkie, M. A., Elliott, J., and Lau, E. (1982) Cancer Res., 42, 1020–1025.PubMedGoogle Scholar
  27. 27.
    Kamisaki, Y., Wada, H., Yagura, T., Nishimura, H., Matsushima, A., and Inada, Y. (1982) Gann, 73, 470–474.PubMedGoogle Scholar
  28. 28.
    Derst, C., Henseling, J., and Rohm, K. H. (2000) Protein Sci., 9, 2009–2017.CrossRefPubMedGoogle Scholar
  29. 29.
    Kotzia, G. A., and Labrou, N. E. (2009) FEBS J., 276, 1750–1761.CrossRefPubMedGoogle Scholar
  30. 30.
    Curran, M. P., Daniel, R. M., Guy, G. R., and Morgan, H. W. (1985) Arch. Biochem. Biophys., 241, 571–576.CrossRefPubMedGoogle Scholar
  31. 31.
    Pritsa, A. A., and Kyriakidis, D. A. (2001) Mol. Cell. Biochem., 216, 93–101.CrossRefPubMedGoogle Scholar
  32. 32.
    Triantafillou, D. J., Georgatsos, J. G., and Kyriakidis, D. A. (1988) Mol. Cell. Biochem., 81, 43–51.CrossRefPubMedGoogle Scholar
  33. 33.
    Yao, M., Yasutake, Y., Morita, H., and Tanaka, I. (2005) Acta Crystallogr. Sec. D Biol. Crystallogr., 61, 294–301.CrossRefGoogle Scholar
  34. 34.
    Bradford, M. M. (1976) Anal. Biochem., 72, 248–254.CrossRefPubMedGoogle Scholar
  35. 35.
    Wriston, J. C., Jr. (1985) Meth. Enzymol., 113, 608–618.CrossRefPubMedGoogle Scholar
  36. 36.
    Ylikangas, P., and Mononen, I. (2000) Anal. Biochem., 280, 42–45.CrossRefPubMedGoogle Scholar
  37. 37.
    Mononen, I., Mononen, T., Ylikangas, P., Kaartinen, V., and Savolainen, K. (1994) Clin. Chem., 40, 385–388.PubMedGoogle Scholar
  38. 38.
    Haki, G. D., and Rakshit, S. K. (2003) Bioresour. Technol., 89, 17–34.CrossRefPubMedGoogle Scholar
  39. 39.
    Ludlow, J. M., and Clark, D. S. (1991) Crit. Rev. Biotechnol., 10, 321–345.CrossRefPubMedGoogle Scholar
  40. 40.
    Cappelletti, D., Chiarelli, L. R., Pasquetto, M. V., Stivala, S., Valentini, G., and Scotti, C. (2008) Biochem. Biophys. Res. Commun., 377, 1222–1226.CrossRefPubMedGoogle Scholar
  41. 41.
    Aung, H. P., Bocola, M., Schleper, S., and Rohm, K. H. (2000) Biochim. Biophys. Acta, 1481, 349–359.PubMedGoogle Scholar
  42. 42.
    Derst, C., Wehner, A., Specht, V., and Rohm, K. H. (1994) Eur. J. Biochem., 224, 533–540.CrossRefPubMedGoogle Scholar

Copyright information

© Pleiades Publishing, Ltd. 2010

Authors and Affiliations

  • S. Bansal
    • 1
  • D. Gnaneswari
    • 1
  • P. Mishra
    • 1
  • B. Kundu
    • 1
    • 2
    Email author
  1. 1.Department of Biochemical Engineering and BiotechnologyIndian Institute of Technology DelhiHauz Khas, New DelhiIndia
  2. 2.School of Biological SciencesIndian Institute of Technology DelhiHauz Khas, New DelhiIndia

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