Establishment of Aromatic Pairs at the Surface of Chondroitinase ABC I: the Effect on Activity and Stability
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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.
KeywordsAromatic pair Chondroitinase ABC I Catalytic efficiency Circular dichroism Fluorometric assay Limited trypsinolysis
This work was supported by the Research Council of Tehran University of Medical Sciences (Grant No. 25021).
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Conflict of Interest
The authors declare that they have no conflict of interest.
- 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.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
- 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
- 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
- 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
- 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
- 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.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
- 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
- 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
- 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
- 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
- 35.Asghari SM (2010) Remarkable improvements of a neutral protease activity and stability share the same structural origins.Google Scholar