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Applied Biochemistry and Biotechnology

, Volume 186, Issue 2, pp 358–370 | Cite as

Establishment of Aromatic Pairs at the Surface of Chondroitinase ABC I: the Effect on Activity and Stability

  • Mohammad Esmaeil Shahaboddin
  • Khosro Khajeh
  • Abolfazl Golestani
Article
  • 115 Downloads

Abstract

Removal of chondroitin sulfate glycosaminoglycan (GAG) chains with chondroitinase ABC I (chABC I) in CNS injury models promotes both saxon regeneration and plasticity. It has been suggested that direct interaction between an aromatic pair appears to contribute about − 1.3 kcal/mol to the stability of a folded protein, so introducing an aromatic pair by point mutation might increase the enzyme activity and thermal stability as in the case of mesophilic xylanase, although using this approach destabilized T4 lysozyme. In this study, we used site-directed mutagenesis to investigate the effect of new aromatic pairs on activity and stability of chABC I. We replaced Ile295, Ser581, and Gly730 adjacent to pre-existing aromatic residues with Tyr to obtain new aromatic pairs, i.e., Tyr295/His372, Tyr576/Tyr581, and Tyr623/Tyr730. Results showed that Km values of S581Y and G730Y variants decreased relative to wild-type enzyme while their catalytic efficiency (kcat/Km) increased but I295Y variant was inactive. Also, long-term and thermal stability of the active mutants was decreased. Fluorescence and circular dichroism studies showed that these mutations resulted in a more flexible enzyme structures: a finding which was confirmed by thermal and limited proteolytic studies. In conclusion, the activity of chABC I can be improved by introducing appropriate aromatic pairs at the enzyme surface. This approach did not provide any promising results regarding the enzyme stability.

Keywords

Aromatic pair Chondroitinase ABC I Catalytic efficiency Circular dichroism Fluorometric assay Limited trypsinolysis 

Notes

Funding Information

This work was supported by the Research Council of Tehran University of Medical Sciences (Grant No. 25021).

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

References

  1. 1.
    Prabhakar, V., Capila, I., Bosques Carlos, J., Pojasek, K., & Sasisekharan, R. (2005). Chondroitinase ABC I from Proteus vulgaris: cloning, recombinant expression and active site identification. Biochemical Journal, 386(1), 103–112.  https://doi.org/10.1042/bj20041222.CrossRefGoogle Scholar
  2. 2.
    Tester, N. J., Plaas, A. H., & Howland, D. R. (2007). Effect of body temperature on chondroitinase ABC’s ability to cleave chondroitin sulfate glycosaminoglycans. Journal of Neuroscience Research, 85(5), 1110–1118.  https://doi.org/10.1002/jnr.21199.CrossRefGoogle Scholar
  3. 3.
    Huang, W., Lunin, V. V., Li, Y., Suzuki, S., Sugiura, N., Miyazono, H., & Cygler, M. (2003). Crystal structure of Proteus vulgaris chondroitin sulfate ABC lyase I at 1.9A resolution. Journal of Molecular Biology, 328(3), 623–634.CrossRefGoogle Scholar
  4. 4.
    Nazari-Robati, M., Khajeh, K., Aminian, M., Mollania, N., & Golestani, A. (2013). Enhancement of thermal stability of chondroitinase ABC I by site-directed mutagenesis: an insight from Ramachandran plot. Biochimica et Biophysica Acta, 1834(2), 479–486.  https://doi.org/10.1016/j.bbapap.2012.11.002.CrossRefGoogle Scholar
  5. 5.
    Hesampour, A., Siadat, S. E., Malboobi, M. A., Mohandesi, N., Arab, S. S., & Ghahremanpour, M. M. (2015). Enhancement of thermostability and kinetic efficiency of Aspergillus niger PhyA phytase by site-directed mutagenesis. Applied Biochemistry and Biotechnology, 175(5), 2528–2541.  https://doi.org/10.1007/s12010-014-1440-y.CrossRefGoogle Scholar
  6. 6.
    Mohammadi, M., Kashi, M. A., Zareian, S., Mirshahi, M., & Khajeh, K. (2014). Remarkable improvement of methylglyoxal synthase thermostability by His-His interaction. Applied Biochemistry and Biotechnology, 172(1), 157–167.  https://doi.org/10.1007/s12010-013-0404-y.CrossRefGoogle Scholar
  7. 7.
    Xu, W., Shao, R., Wang, Z., & Yan, X. (2015). Improving the neutral phytase activity from Bacillus amyloliquefaciens DSM 1061 by site-directed mutagenesis. Applied Biochemistry and Biotechnology, 175(6), 3184–3194.  https://doi.org/10.1007/s12010-015-1495-4.CrossRefGoogle Scholar
  8. 8.
    Kannan, N., & Vishveshwara, S. (2000). Aromatic clusters: a determinant of thermal stability of thermophilic proteins. Protein Engineering, 13(11), 753–761.CrossRefGoogle Scholar
  9. 9.
    Goldstein, R. A. (2007). Amino-acid interactions in psychrophiles, mesophiles, thermophiles, and hyperthermophiles: insights from the quasi-chemical approximation. Protein Science : a Publication of the Protein Society, 16(9), 1887–1895.  https://doi.org/10.1110/ps.072947007.CrossRefGoogle Scholar
  10. 10.
    Serrano, L., Bycroft, M., & Fersht, A. R. (1991). Aromatic-aromatic interactions and protein stability. Investigation by double-mutant cycles. Journal of Molecular Biology, 218(2), 465–475.CrossRefGoogle Scholar
  11. 11.
    Burley, S. K., & Petsko, G. A. (1985). Aromatic-aromatic interaction: a mechanism of protein structure stabilization. Science (New York, N.Y.), 229(4708), 23–28.CrossRefGoogle Scholar
  12. 12.
    Georis, J., de Lemos Esteves, F., Lamotte-Brasseur, J., Bougnet, V., Devreese, B., Giannotta, F., Granier, B., & Frere, J. M. (2000). An additional aromatic interaction improves the thermostability and thermophilicity of a mesophilic family 11 xylanase: structural basis and molecular study. Protein Science : a Publication of the Protein Society, 9(3), 466–475.  https://doi.org/10.1110/ps.9.3.466.CrossRefGoogle Scholar
  13. 13.
    Mooers, B. H., Baase, W. A., Wray, J. W., & Matthews, B. W. (2009). Contributions of all 20 amino acids at site 96 to the stability and structure of T4 lysozyme. Protein Science : a Publication of the Protein Society, 18(5), 871–880.  https://doi.org/10.1002/pro.94.CrossRefGoogle Scholar
  14. 14.
    Fisher, C. L., & Pei, G. K. (1997). Modification of a PCR-based site-directed mutagenesis method. BioTechniques, 23(4), 570–571 574.CrossRefGoogle Scholar
  15. 15.
    Fan, X. J., Yang, C., Zhang, C., Ren, H., & Zhang, J. D. (2018). Cloning, site-directed mutagenesis, and functional analysis of active residues in Lymantria dispar Chitinase. Applied Biochemistry and Biotechnology, 184(1), 12–24.  https://doi.org/10.1007/s12010-017-2524-2.CrossRefGoogle Scholar
  16. 16.
    Goomber, S., Kumar, A., Singh, R., & Kaur, J. (2016). Point mutation Ile137-met near surface conferred psychrophilic behaviour and improved catalytic efficiency to Bacillus lipase of 1.4 subfamily. Applied Biochemistry and Biotechnology, 178(4), 753–765.  https://doi.org/10.1007/s12010-015-1907-5.CrossRefGoogle Scholar
  17. 17.
    Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72(1-2), 248–254.CrossRefGoogle Scholar
  18. 18.
    Shirdel, S. A., Khalifeh, K., Ranjbar, B., Golestani, A., & Khajeh, K. (2016). Unfolding of chondroitinase ABC iota is dependent on thermodynamic driving force by kinetically rate constant-amplitude compensation: A stopped-flow fluorescence study. Enzyme and Microbial Technology, 93-94, 200–206.  https://doi.org/10.1016/j.enzmictec.2016.09.001.CrossRefGoogle Scholar
  19. 19.
    Shaya, D., Hahn, B. S., Bjerkan, T. M., Kim, W. S., Park, N. Y., Sim, J. S., Kim, Y. S., & Cygler, M. (2008). Composite active site of chondroitin lyase ABC accepting both epimers of uronic acid. Glycobiology, 18(3), 270–277.  https://doi.org/10.1093/glycob/cwn002.CrossRefGoogle Scholar
  20. 20.
    Tomazic, S. J., & Klibanov, A. M. (1988). Mechanisms of irreversible thermal inactivation of Bacillus alpha-amylases. The Journal of Biological Chemistry, 263(7), 3086–3091.Google Scholar
  21. 21.
    Nazari-Robati, M., Golestani, A., & Asadikaram, G. (2016). Improvement of proteolytic and oxidative stability of Chondroitinase ABC I by cosolvents. International Journal of Biological Macromolecules, 91, 812–817.  https://doi.org/10.1016/j.ijbiomac.2016.06.030.CrossRefGoogle Scholar
  22. 22.
    Bradbury, E. J., & Carter, L. M. (2011). Manipulating the glial scar: chondroitinase ABC as a therapy for spinal cord injury. Brain Research Bulletin, 84(4–5), 306–316.  https://doi.org/10.1016/j.brainresbull.2010.06.015.CrossRefGoogle Scholar
  23. 23.
    Nazari-Robati, M., Khajeh, K., Aminian, M., Fathi-Roudsari, M., & Golestani, A. (2012). Co-solvent mediated thermal stabilization of chondroitinase ABC I form Proteus vulgaris. International Journal of Biological Macromolecules, 50(3), 487–492.  https://doi.org/10.1016/j.ijbiomac.2012.01.009.CrossRefGoogle Scholar
  24. 24.
    Bagherieh, M., Kheirollahi, A., Shahaboddin, M. E., Khajeh, K., & Golestani, A. (2017). Calcium and TNFalpha additively affect the chondroitinase ABC I activity. International Journal of Biological Macromolecules, 103, 1201–1206.  https://doi.org/10.1016/j.ijbiomac.2017.05.177.CrossRefGoogle Scholar
  25. 25.
    Shahaboddin, M. E., Khajeh, K., Maleki, M., & Golestani, A. (2017). Improvement of activity and stability of Chondroitinase ABC I by introducing an aromatic cluster at the surface of protein. Enzyme and Microbial Technology, 105, 38–44.  https://doi.org/10.1016/j.enzmictec.2017.06.004.CrossRefGoogle Scholar
  26. 26.
    Kheirollahi, A., Khajeh, K., & Golestani, A. (2017). Rigidifying flexible sites: An approach to improve stability of chondroitinase ABC I. International Journal of Biological Macromolecules, 97, 270–278.  https://doi.org/10.1016/j.ijbiomac.2017.01.027.CrossRefGoogle Scholar
  27. 27.
    Akram Shirdel, S., Khalifeh, K., Golestani, A., Ranjbar, B., & Khajeh, K. (2015). Critical role of a loop at C-terminal domain on the conformational stability and catalytic efficiency of Chondroitinase ABC I. Molecular Biotechnology, 57(8), 727–734.  https://doi.org/10.1007/s12033-015-9864-3.CrossRefGoogle Scholar
  28. 28.
    Suzuki, T., Akimoto, M., Imai, H., Ueda, Y., Mandai, M., Yoshimura, N., Swaroop, A., & Takahashi, M. (2007). Chondroitinase ABC treatment enhances synaptogenesis between transplant and host neurons in model of retinal degeneration. Cell Transplantation, 16(5), 493–503.CrossRefGoogle Scholar
  29. 29.
    Jaenicke, R. (1991). Protein stability and molecular adaptation to extreme conditions. European Journal of Biochemistry, 202(3), 715–728.CrossRefGoogle Scholar
  30. 30.
    Somero, G. N. (1995). Proteins and temperature. Annual Review of Physiology, 57(1), 43–68.  https://doi.org/10.1146/annurev.ph.57.030195.000355.CrossRefGoogle Scholar
  31. 31.
    Teilum, K., Olsen, J. G., & Kragelund, B. B. (2009). Functional aspects of protein flexibility. Cellular and Molecular Life Sciences : CMLS, 66(14), 2231–2247.  https://doi.org/10.1007/s00018-009-0014-6.CrossRefGoogle Scholar
  32. 32.
    Teilum, K., Olsen, J. G., & Kragelund, B. B. (2011). Protein stability, flexibility and function. Biochimica et Biophysica Acta, 1814(8), 969–976.  https://doi.org/10.1016/j.bbapap.2010.11.005.CrossRefGoogle Scholar
  33. 33.
    Shamsi, M., Shirdel, S. A., Jafarian, V., Jafari, S. S., Khalifeh, K., & Golestani, A. (2016). Optimization of conformational stability and catalytic efficiency in chondroitinase ABC Ι by protein engineering methods. Engineering in Life Sciences, 16(8), 690–696.  https://doi.org/10.1002/elsc.201600034.CrossRefGoogle Scholar
  34. 34.
    Lo Conte, L., Ailey, B., Hubbard, T. J., Brenner, S. E., Murzin, A. G., & Chothia, C. (2000). SCOP: a structural classification of proteins database. Nucleic Acids Research, 28(1), 257–259.CrossRefGoogle Scholar
  35. 35.
    Asghari SM (2010) Remarkable improvements of a neutral protease activity and stability share the same structural origins.Google Scholar
  36. 36.
    Eftink, M. R., & Ghiron, C. A. (1976). Exposure of tryptophanyl residues in proteins. Quantitative determination by fluorescence quenching studies. Biochemistry, 15(3), 672–680.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Research Center for Biochemistry and Nutrition in Metabolic DiseasesKashan University of Medical SciencesKashanIran
  2. 2.Department of Biochemistry, Faculty of Biological SciencesTarbiat Modares UniversityTehranIran
  3. 3.Department of Clinical Biochemistry, School of MedicineTehran University of Medical SciencesTehranIran

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