Skip to main content

Unscrambling the Effect of C-Terminal Tail Deletion on the Stability of a Cold-Adapted, Organic Solvent Stable Lipase from Staphylococcus epidermidis AT2

Molecular Biotechnology volume 56, pages 747–757 (2014)Cite this article

Abstract

Terminal moieties of most proteins are long known to be disordered and flexible. To unravel the functional role of these regions on the structural stability and biochemical properties of AT2 lipase, four C-terminal end residues, (Ile–Thr–Arg–Lys) which formed a flexible, short tail-like random-coil segment were targeted for mutation. Swapping of the tail-like region had resulted in an improved crystallizability and anti-aggregation property along with a slight shift of the thermostability profile. The lipolytic activity of mutant (M386) retained by 43 % compared to its wild-type with 18 % of the remaining activity at 45 °C. In silico analysis conducted at 25 and 45 °C was found to be in accordance to the experimental findings in which the RMSD values of M386 were more stable throughout the total trajectory in comparison to its wild-type. Terminal moieties were also observed to exhibit large movement and flexibility as denoted by high RMSF values at both dynamics. Variation in organic solvent stability property was detected in M386 where the lipolytic activity was stimulated in the presence of 25 % (v/v) of DMSO, isopropanol, and diethyl ether. This may be worth due to changes in the surface charge residues at the mutation point which probably involve in protein–solvent interaction.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

43,34 €

Price includes VAT (Finland)
Tax calculation will be finalised during checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

References

  1. Dunker, A. K., Lawson, J. D., Brown, C. J., Williams, R. M., Romero, P., et al. (2001). Intrinsically disordered protein. Journal of Molecular Graphics and Modelling, 19, 26–59.

    CAS  Article  Google Scholar 

  2. Joseph, B., Ramteke, P. W., & Thomas, G. (2008). Cold active microbial lipases: Some hot issues and recent developments. Biotechnology Advances, 26, 457–470.

    CAS  Article  Google Scholar 

  3. Li, W. F., Zhou, X. X., & Lu, P. (2005). Structural features of thermozymes. Biotechnology Advances, 23, 271–281.

    CAS  Article  Google Scholar 

  4. Dale, G. E., Oefner, C., & D’Arcy, A. (2003). The protein as a variable in protein crystallization. Journal of Structural Biology, 142, 88–97.

    CAS  Article  Google Scholar 

  5. Alexandrov, N. (1993). Structural argument for N-terminal initiation of protein folding. Protein Science, 2, 1989–1991.

    CAS  Article  Google Scholar 

  6. Sharma, S., Zheng, H., Huang, Y. J., Ertekin, A., Hamuro, Y., et al. (2009). Construct optimization for protein NMR structure analysis using amide hydrogen/deuterium exchange mass spectrometry. Proteins, 76, 882–894.

    CAS  Article  Google Scholar 

  7. Fernando, P., Abdulle, R., Mohindra, A., Guillemette, J. G., & Heikkila, J. J. (2002). Mutation or deletion of the C-terminal tail affects the function and structure of Xenopus laevis small heat shock protein, hsp30. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 133, 95–103.

    CAS  Article  Google Scholar 

  8. Martin, G., Keller, W., & Doublié, S. (2000). Crystal structure of mammalian poly (A) polymerase in complex with an analog of ATP. EMBO Journal, 19, 4193–4203.

    CAS  Article  Google Scholar 

  9. Rahman, R. N. Z. R. A., Kamarudin, N. H. A., Yunus, J., Salleh, A. B., & Basri, M. (2010). Expression of an organic solvent stable lipase from Staphylococcus epidermidis AT2. International Journal of Molecular Sciences, 11, 3195–3208.

    Article  Google Scholar 

  10. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685.

    CAS  Article  Google Scholar 

  11. Kwon, D. Y., & Rhee, J. S. (1986). A simple and rapid colorimetric method for determination of free fatty acids for lipase assay. Journal of the American Oil Chemist’s Society, 63, 89–92.

    CAS  Article  Google Scholar 

  12. Krieger, E., Koraimann, G., & Vriend, G. (2002). Increasing the precision of comparative models with YASARA NOVA—A self-parameterizing force field. Proteins, 47, 393–402.

    CAS  Article  Google Scholar 

  13. Krieger, E., Darden, T., Nabuurs, S., Finkelstein, A., & Vriend, G. (2004). Making optimal use of empirical energy functions: Force-field parameterization in crystal space. Proteins, 57, 678–683.

    CAS  Article  Google Scholar 

  14. Noble, M. E., Cleasby, A., Johnson, L. N., Egmond, M. R., & Frenken, L. G. (1993). The crystal structure of triacylglycerol lipase from Pseudomonas glumae reveals a partially redundant catalytic aspartate. FEBS Letters, 331, 123–128.

    CAS  Article  Google Scholar 

  15. Tyndall, J. D. A., Sinchaikul, S., Fothergill-Gilmore, L. A., Taylor, P., & Walkinshaw, M. D. (2002). Crystal structure of a thermostable lipase from Bacillus stearothermophilus P1. Journal of Molecular Biology, 323, 859–869.

    CAS  Article  Google Scholar 

  16. Tiesinga, J. J. W., Pauderoyan, G. V., Nardini, M., Ransac, S., & Dijkstra, B. W. (2007). Structural basis of phospholipase activity of Staphylococcus hyicus lipase. Journal of Molecular Biology, 371, 447–456.

    CAS  Article  Google Scholar 

  17. Arakawa, T., Philo, J. S., & Kita, Y. (2001). Kinetic and thermodynamic analysis of thermal unfolding of recombinant erythropoietin. Bioscience, Biotechnology, and Biochemistry, 65, 1321–1327.

    CAS  Article  Google Scholar 

  18. Nybo, K. (2012). Troubleshooting forum, molecular biology and techniques: Native PAGE. BioTechniques, 52, 20–21.

    Google Scholar 

  19. Gu, J., & Hilser, V. (2009). The significance and impacts of protein disorder and conformational variants. In J. Gu & P. E. Bourne (Eds.), Structural bioinformatics (pp. 939–962). Hoboken: Wiley.

    Google Scholar 

  20. Buske, P. J., & Levin, P. A. (2013). A flexible C-terminal linker is required for proper FtsZ assembly in vitro and cytokinetic ring formation in vivo. Molecular Microbiology, 89, 249–263.

    CAS  Article  Google Scholar 

  21. Liao, J. H., Lee, J. S., Wu, S. H., & Chiou, S. H. (2009). COOH-terminal truncations and site-directed mutations enhance thermostability and chaperone-like activity of porcine αB-crystallin. Molecular Vision, 15, 1429–1444.

    CAS  Google Scholar 

  22. McPherson, A. (2001). A comparison of salts for the crystallization of macromolecules. Protein Science, 10, 418–422.

    CAS  Article  Google Scholar 

  23. Thornton, J. M., & Sibanda, B. L. (1983). Amino and carboxy-terminal regions in globular proteins. Journal of Molecular Biology, 167, 443–460.

    CAS  Article  Google Scholar 

  24. Derewenda, Z. S. (2010). Application of protein engineering to enhance protein crystallizability and improve crystal properties. Acta Crystallographica Section D, 66, 604–615.

    CAS  Article  Google Scholar 

  25. Evdokimov, A. G., Pokross, M., Walter, R., Mekel, M., Cox, B., et al. (2006). Engineering the catalytic domain of human protein tyrosine phosphatase β for structure-based drug discovery. Acta Crystallographica Section D, 62, 1435–1445.

    CAS  Article  Google Scholar 

  26. Russel, R. J. M., Ferguson, J. M. C., Hough, D. W., Danson, M. J., & Taylor, G. L. (1997). The crystal structure of citrate synthase from hyperthermophilic archeon Pyrococcus furiosus at 1.9 Å resolution. Biochemistry, 36, 9983–9994.

    Article  Google Scholar 

  27. Liu, L., Zhang, G., Zhang, Z., Wang, S., & Chen, H. (2011). Terminal amino acids disturb xylanase thermostability and activity. Journal of Biological Chemistry, 286, 44710–44715.

    CAS  Article  Google Scholar 

  28. Haran, G., Haas, E., & Rapaport, D. C. (1994). Molecular dynamics simulations of simple peptide models: Solvent effects and comparison with experiment. The Journal of Physical Chemistry, 98, 10294–10302.

    CAS  Article  Google Scholar 

  29. Karjiban, R. A., Rahman, M. B. A., Salleh, A. B., Basri, M., Rahman, R. N. Z. R. A., & Chor, A. L. T. (2009). On the importance of the small domain in the thermostability of thermoalkalophilic lipases from L1 and T1: Insights from molecular dynamics simulation. Protein and Peptide Letters, 17, 699–707.

    Article  Google Scholar 

  30. Kamal, M. Z., Mohammad, T. A. S., Krishnamoorthy, G., & Rao, N. M. (2012). Role of active site rigidity in activity: MD simulation and fluorescence study on a lipase mutant. PLoS ONE, 7, 1–8.

    Article  Google Scholar 

  31. Iwakura, M., & Honda, S. (1996). Stability and reversibility of thermal denaturation are greatly improved by limiting terminal flexibility of Escherichia coli dihydrofolate reductase. Journal of Biochemistry, 119, 414–420.

    CAS  Article  Google Scholar 

  32. Salleh, A. B., Abd Rahim, A. S. M., Abdul Rahman, R. N. Z. R., Leow, T. C., & Basri, M. (2012). The role of Arg157Ser in improving the compactness and stability of ARM lipase. Journal of Computational Science and System Biology, 5, 039–046.

    CAS  Google Scholar 

  33. Chakravorty, D., Parameswaran, S., Dubey, V. K., & Patra, S. (2012). Unraveling the rationale behind organic solvent stability of lipases. Applied Biochemistry and Biotechnology, 167, 439–461.

    CAS  Article  Google Scholar 

  34. Laane, C., Boeren, S., Vos, K., & Veeger, C. (1987). Rules for optimization of biocatalysis in organic solvents. Biotechnology and Bioengineering, 30, 81–87.

Download references

Acknowledgments

This project was financially supported by Ministry of Science and Technology (MOSTI), Malaysia (10-01-04-SS07).

Author information

Affiliations

  1. Enzyme and Microbial Technology Research Centre, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia

    Nor Hafizah Ahmad Kamarudin, Raja Noor Zaliha Raja Abd Rahman, Mohd Shukuri Mohamad Ali, Thean Chor Leow, Mahiran Basri & Abu Bakar Salleh

  2. Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia

    Nor Hafizah Ahmad Kamarudin, Raja Noor Zaliha Raja Abd Rahman, Mohd Shukuri Mohamad Ali, Thean Chor Leow & Abu Bakar Salleh

  3. Faculty of Science, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia

    Mahiran Basri

Authors
  1. Nor Hafizah Ahmad Kamarudin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Raja Noor Zaliha Raja Abd Rahman
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mohd Shukuri Mohamad Ali
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Thean Chor Leow
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Mahiran Basri
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Abu Bakar Salleh
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Raja Noor Zaliha Raja Abd Rahman.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Kamarudin, N.H.A., Rahman, R.N.Z.R.A., Ali, M.S.M. et al. Unscrambling the Effect of C-Terminal Tail Deletion on the Stability of a Cold-Adapted, Organic Solvent Stable Lipase from Staphylococcus epidermidis AT2. Mol Biotechnol 56, 747–757 (2014). https://doi.org/10.1007/s12033-014-9753-1

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-014-9753-1

Keywords

  • C-terminal region
  • Deletion
  • Crystallizability
  • Thermostability
  • Organic solvent stability