Molecular Biotechnology

, Volume 60, Issue 9, pp 698–711 | Cite as

Ig-like Domain in Endoglucanase Cel9A from Alicyclobacillus acidocaldarius Makes Dependent the Enzyme Stability on Calcium

  • Mohammad PazhangEmail author
  • Fereshteh S. Younesi
  • Faramarz Mehrnejad
  • Saeed Najavand
  • Alireza Tarinejad
  • Mehrnaz Haghi
  • Fatemeh Rashno
  • Khosro Khajeh
Original Paper


Endoglucanase Cel9A from Alicyclobacillus acidocaldarius (AaCel9A) has an Ig-like domain and the enzyme stability is dependent to calcium. In this study the effect of calcium on the structure and stability of the wild-type enzyme and the truncated form (the wild-type enzyme without Ig-like domain, AaCel9AΔN) was investigated. Fluorescence quenching results indicated that calcium increased and decreased the rigidity of the wild-type and truncated enzymes, respectively. RMSF results indicated that AaCel9A has two flexible regions (regions A and B) and deleting the Ig-like domain increased the truncated enzyme stability by decreasing the flexibility of region B probably through increasing the hydrogen bonds. Calcium contact map analysis showed that deleting the Ig-like domain decreased the calcium contacting residues and their calcium binding affinities, especially, in region B which has a role in calcium binding site in AaCel9A. Metal depletion and activity recovering as well as stability results showed that the structure and stability of the wild-type and truncated enzymes are completely dependent on and independent of calcium, respectively. Finally, one can conclude that the deletion of Ig-like domain makes AaCel9AΔN independent of calcium via decreasing the flexibility of region B through increasing the hydrogen bonds. This suggests a new role for the Ig-like domain which makes AaCel9A structure dependent on calcium.


Endoglucanase Cel9A Ig-like domain Molecular dynamics simulation Calcium 



Alicyclobacillus acidocaldarius endoglucanase Cel9A


AaCel9A without Ig-like domain


Carbohydrate binding domain




Molecular dynamics


Radial distribution function


Root-mean-square fluctuation



The authors express their gratitude to the research council of Azarbaijan Shahid Madani University for the financial support during the course of this project.

Compliance with Ethical Standards

Conflict of interest

The authors declare that there are no conflicts of interest.


  1. 1.
    Liao, J. C., Mi, L., Pontrelli, S., & Luo, S. (2016). Fuelling the future: Microbial engineering for the production of sustainable biofuels. Nature Reviews Microbiology, 14, 288–304.CrossRefPubMedGoogle Scholar
  2. 2.
    Mussatto, S. I., Dragone, G., Guimarães, P. M., Silva, J. P. A., Carneiro, L. M., Roberto, I. C., Vicente, A., Domingues, L., & Teixeira, J. A. (2010). Technological trends, global market, and challenges of bio-ethanol production. Biotechnology Advances, 28, 817–830.CrossRefPubMedGoogle Scholar
  3. 3.
    Lin, C., Shen, Z., & Qin, W. (2017). Characterization of xylanase and cellulase produced by a newly isolated Aspergillus fumigatus N2 and its efficient saccharification of Barley Straw. Applied Biochemistry and Biotechnology, 182, 559–569.CrossRefPubMedGoogle Scholar
  4. 4.
    Cao, Y., & Tan, H. (2004). Structural characterization of cellulose with enzymatic treatment. Journal of Molecular Structure, 705, 189–193.CrossRefGoogle Scholar
  5. 5.
    Bayer, E. A., Chanzy, H., Lamed, R., & Shoham, Y. (1998). Cellulose, cellulases and cellulosomes. Current Opinion in Structural Biology, 8, 548–557.CrossRefPubMedGoogle Scholar
  6. 6.
    Knowles, J., Lehtovaara, P., & Teeri, T. (1987). Cellulase families and their genes. Trends in Biotechnology, 5, 255–261.CrossRefGoogle Scholar
  7. 7.
    Zhang, Y.-H. P., Himmel, M. E., & Mielenz, J. R. (2006). Outlook for cellulase improvement: Screening and selection strategies. Biotechnology Advances, 24, 452–481.CrossRefGoogle Scholar
  8. 8.
    Wilson, D. B., & Irwin, D. C. (1999). Genetics and properties of cellulases. In G. T. Tsao, et al. (Eds.), Recent progress in bioconversion of lignocellulosics. Advances in biochemical engineering/biotechnology (Vol. 65). Berlin, Heidelberg: Springer.Google Scholar
  9. 9.
    Berka, R. M., Grigoriev, I. V., Otillar, R., Salamov, A., Grimwood, J., Reid, I., Ishmael, N., John, T., Darmond, C., & Moisan, M.-C. (2011). Comparative genomic analysis of the thermophilic biomass-degrading fungi Myceliophthora thermophila and Thielavia terrestris. Nature Biotechnology, 29, 922–927.CrossRefPubMedGoogle Scholar
  10. 10.
    Bhalla, A., Bansal, N., Kumar, S., Bischoff, K. M., & Sani, R. K. (2013). Improved lignocellulose conversion to biofuels with thermophilic bacteria and thermostable enzymes. Bioresource Technology, 128, 751–759.CrossRefPubMedGoogle Scholar
  11. 11.
    Dick, M., Weiergräber, O. H., Classen, T., Bisterfeld, C., Bramski, J., Gohlke, H., & Pietruszka, J. (2016). Trading off stability against activity in extremophilic aldolases. Scientific Reports, 6, 17908.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Kim, S. J., Joo, J. E., Jeon, S. D., Hyeon, J. E., Kim, S. W., Um, Y. S., & Han, S. O. (2016). Enhanced thermostability of mesophilic endoglucanase Z with a high catalytic activity at active temperatures. International Journal of Biological Macromolecules, 86, 269–276.CrossRefPubMedGoogle Scholar
  13. 13.
    Moran-Mirabal, J. M., Bolewski, J. C., & Walker, L. P. (2011). Reversibility and binding kinetics of Thermobifida fusca cellulases studied through fluorescence recovery after photobleaching microscopy. Biophysical Chemistry, 155, 20–28.CrossRefPubMedGoogle Scholar
  14. 14.
    Eckert, K., Zielinski, F., Lo Leggio, L., & Schneider, E. (2002). Gene cloning, sequencing, and characterization of a family 9 endoglucanase (CelA) with an unusual pattern of activity from the thermoacidophile Alicyclobacillus acidocaldarius ATCC27009. Applied Microbiology and Biotechnology, 60, 428–436.CrossRefPubMedGoogle Scholar
  15. 15.
    Pereira, J. H., Sapra, R., Volponi, J. V., Kozina, C. L., Simmons, B., & Adams, P. D. (2009). Structure of endoglucanase Cel9A from the thermoacidophilic Alicyclobacillus acidocaldarius. Acta Crystallographica Section D: Biological Crystallography, 65, 744–750.CrossRefGoogle Scholar
  16. 16.
    Eckert, K., Vigouroux, A., Leggio, L. L., & Moréra, S. (2009). Crystal structures of A. acidocaldarius endoglucanase Cel9A in complex with cello-oligosaccharides: Strong -1 and -2 subsites mimic cellobiohydrolase activity. Journal of Molecular Biology, 394, 61–70.CrossRefPubMedGoogle Scholar
  17. 17.
    Wang, H.-J., Hsiao, Y.-Y., Chen, Y.-P., Ma, T.-Y., & Tseng, C.-P. (2016). Polarity alteration of a calcium site induces a hydrophobic interaction network and enhances Cel9A endoglucanase thermostability. Applied and Environmental Microbiology, 82, 1662–1674.CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Liu, H., Pereira, J. H., Adams, P. D., Sapra, R., Simmons, B. A., & Sale, K. L. (2010). Molecular simulations provide new insights into the role of the accessory immunoglobulin-like domain of Cel9A. FEBS Letters, 584, 3431–3435.CrossRefPubMedGoogle Scholar
  19. 19.
    Younesi, F. S., Pazhang, M., Najavand, S., Rahimizadeh, P., Akbarian, M., Mohammadian, M., & Khajeh, K. (2016). Deleting the Ig-like domain of Alicyclobacillus acidocaldarius endoglucanase Cel9A causes a simultaneous increase in the activity and stability. Molecular Biotechnology, 58, 12–21.CrossRefPubMedGoogle Scholar
  20. 20.
    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, 248–254.CrossRefPubMedGoogle Scholar
  21. 21.
    Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry, 193, 265–275.PubMedGoogle Scholar
  22. 22.
    Van Der Spoel, D., Lindahl, E., Hess, B., Groenhof, G., Mark, A. E., & Berendsen, H. J. (2005). GROMACS: Fast, flexible, and free. Journal of Computational Chemistry, 26, 1701–1718.CrossRefGoogle Scholar
  23. 23.
    Hess, B., Kutzner, C., Van Der Spoel, D., & Lindahl, E. (2008). GROMACS 4: Algorithms for highly efficient, load-balanced, and scalable molecular simulation. Journal of Chemical Theory and Computation, 4, 435–447.CrossRefPubMedGoogle Scholar
  24. 24.
    Oostenbrink, C., Villa, A., Mark, A. E., & Van Gunsteren, W. F. (2004). A biomolecular force field based on the free enthalpy of hydration and solvation: The GROMOS force-field parameter sets 53A5 and 53A6. Journal of Computational Chemistry, 25, 1656–1676.CrossRefPubMedGoogle Scholar
  25. 25.
    Berendsen, H., Grigera, J., & Straatsma, T. (1987). The missing term in effective pair potentials. Journal of Physical Chemistry, 91, 6269–6271.CrossRefGoogle Scholar
  26. 26.
    Berendsen, H. J., van der Spoel, D., & van Drunen, R. (1995). GROMACS: A message-passing parallel molecular dynamics implementation. Computer Physics Communications, 91, 43–56.CrossRefGoogle Scholar
  27. 27.
    Hess, B., Bekker, H., Berendsen, H. J., & Fraaije, J. G. (1997). LINCS: A linear constraint solver for molecular simulations. Journal of Computational Chemistry, 18, 1463–1472.CrossRefGoogle Scholar
  28. 28.
    Darden, T., York, D., & Pedersen, L. (1993). Particle mesh Ewald: An N·log (N) method for Ewald sums in large systems. The Journal of Chemical Physics, 98, 10089–10092.CrossRefGoogle Scholar
  29. 29.
    Blau, C., & Grubmuller, H. (2013). g_contacts: Fast contact search in bio-molecular ensemble data. Computer Physics Communications, 184, 2856–2859.CrossRefGoogle Scholar
  30. 30.
    Rabinovich, M., Melnick, M., & Bolobova, A. (2002). The structure and mechanism of action of cellulolytic enzymes. Biochemistry, 67, 850–871.PubMedGoogle Scholar
  31. 31.
    Sukharnikov, L. O., Cantwell, B. J., Podar, M., & Zhulin, I. B. (2011). Cellulases: Ambiguous nonhomologous enzymes in a genomic perspective. Trends in Biotechnology, 29, 473–479.CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Cheng, R., Chen, J., Yu, X., Wang, Y., Wang, S., & Zhang, J. (2013). Recombinant production and characterization of full-length and truncated β-1, 3-glucanase PglA from Paenibacillus sp. S09. BMC Biotechnology, 13, 105.CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Kataeva, I. A., Uversky, V. N., Brewer, J. M., Schubot, F., Rose, J. P., Wang, B.-C., & Ljungdahl, L. G. (2004). Interactions between immunoglobulin-like and catalytic modules in Clostridium thermocellum cellulosomal cellobiohydrolase CbhA. Protein Engineering Design and Selection, 17, 759–769.CrossRefGoogle Scholar
  34. 34.
    Han, Q., Liu, N., Robinson, H., Cao, L., Qian, C., Wang, Q., Xie, L., Ding, H., Wang, Q., & Huang, Y. (2013). Biochemical characterization and crystal structure of a GH10 xylanase from termite gut bacteria reveal a novel structural feature and significance of its bacterial Ig-like domain. Biotechnology and Bioengineering, 110, 3093–3103.CrossRefPubMedGoogle Scholar
  35. 35.
    Pazhang, M., Mehrnejad, F., Pazhang, Y., Falahati, H., & Chaparzadeh, N. (2016). Effect of sorbitol and glycerol on the stability of trypsin and difference between their stabilization effects in the various solvents. Biotechnology and Applied Biochemistry, 63, 206–213.CrossRefPubMedGoogle Scholar
  36. 36.
    Andersen, C. A., Palmer, A. G., Brunak, S., & Rost, B. (2002). Continuum secondary structure captures protein flexibility. Structure, 10, 175–184.CrossRefPubMedGoogle Scholar
  37. 37.
    Rashin, A. A., Rashin, A. H., & Jernigan, R. L. (2010). Diversity of function-related conformational changes in proteins: Coordinate uncertainty, fragment rigidity, and stability. Biochemistry, 49, 5683–5704.CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Mamonova, T. B., Glyakina, A. V., Galzitskaya, O. V., & Kurnikova, M. G. (2013). Stability and rigidity/flexibility—Two sides of the same coin? Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics, 1834, 854–866.CrossRefGoogle Scholar
  39. 39.
    Van Der Lee, R., Buljan, M., Lang, B., Weatheritt, R. J., Daughdrill, G. W., Dunker, A. K., Fuxreiter, M., Gough, J., Gsponer, J., & Jones, D. T. (2014). Classification of intrinsically disordered regions and proteins. Chemical Reviews, 114, 6589–6631.CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Hsu, Y.-H., Burke, J. E., Stephens, D. L., Deems, R. A., Li, S., Asmus, K. M., Woods, V. L., & Dennis, E. A. (2008). Calcium binding rigidifies the C2 domain and the intradomain interaction of GIVA phospholipase A2 as revealed by hydrogen/deuterium exchange mass spectrometry. Journal of Biological Chemistry, 283, 9820–9827.CrossRefPubMedGoogle Scholar
  41. 41.
    Chen, A., Li, Y., Nie, J., McNeil, B., Jeffrey, L., Yang, Y., & Bai, Z. (2015). Protein engineering of Bacillus acidopullulyticus pullulanase for enhanced thermostability using in silico data driven rational design methods. Enzyme and Microbial Technology, 78, 74–83.CrossRefPubMedGoogle Scholar
  42. 42.
    Bonito, C. A., Nunes, J., Leandro, J., Louro, F., Leandro, P., Ventura, F. V., & Guedes, R. C. (2016). Unveiling the pathogenic molecular mechanisms of the most common variant (p.K329E) in medium-chain acyl-CoA dehydrogenase deficiency by in vitro and in silico approaches. Biochemistry, 55, 7086–7098.CrossRefPubMedGoogle Scholar
  43. 43.
    Zheng, H., Chruszcz, M., Lasota, P., Lebioda, L., & Minor, W. (2008). Data mining of metal ion environments present in protein structures. Journal of Inorganic Biochemistry, 102, 1765–1776.CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Domínguez, D. C., Guragain, M., & Patrauchan, M. (2015). Calcium binding proteins and calcium signaling in prokaryotes. Cell Calcium, 57, 151–165.CrossRefPubMedGoogle Scholar
  45. 45.
    Kumagai, Y., Kawakami, K., Mukaihara, T., Kimura, M., & Hatanaka, T. (2012). The structural analysis and the role of calcium binding site for thermal stability in mannanase. Biochimie, 94, 2783–2790.CrossRefPubMedGoogle Scholar
  46. 46.
    Lee, S., Park, H. I., & Sang, Q.-X. A. (2007). Calcium regulates tertiary structure and enzymatic activity of human endometase/matrilysin-2 and its role in promoting human breast cancer cell invasion. Biochemical Journal, 403, 31–42.CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Wojcik, M., & Stec, W. J. (2010). The effect of divalent cations on the catalytic activity of the human plasma 3′-exonuclease. BioMetals, 23, 1113–1121.CrossRefPubMedGoogle Scholar
  48. 48.
    Veltman, O. R., Vriend, G., van den Burg, B., Hardy, F., Venema, G., & Eijsink, V. G. (1997). Engineering thermolysin-like proteases whose stability is largely independent of calcium. FEBS Letters, 405, 241–244.CrossRefPubMedGoogle Scholar
  49. 49.
    Bodelon, G., Palomino, C., & Fernandez, L. A. (2013). Immunoglobulin domains in Escherichia coli and other enterobacteria: From pathogenesis to applications in antibody technologies. FEMS Microbiology Reviews, 37, 204–250.CrossRefPubMedGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Mohammad Pazhang
    • 1
    Email author
  • Fereshteh S. Younesi
    • 1
  • Faramarz Mehrnejad
    • 2
  • Saeed Najavand
    • 1
  • Alireza Tarinejad
    • 3
  • Mehrnaz Haghi
    • 1
  • Fatemeh Rashno
    • 4
  • Khosro Khajeh
    • 4
  1. 1.Department of Cellular and Molecular Biology, Faculty of ScienceAzarbaijan Shahid Madani UniversityTabrizIran
  2. 2.Department of Life Science Engineering, Faculty of New Sciences & TechnologiesUniversity of TehranTehranIran
  3. 3.Department of Biotechnology, Faculty of AgricultureAzarbaijan Shahid Madani UniversityTabrizIran
  4. 4.Department of Biochemistry, Faculty of Biological ScienceTarbiat Modares UniversityTehranIran

Personalised recommendations