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Influence of surface charge, binding site residues and glycosylation on Thielavia terrestris cutinase biochemical characteristics

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Abstract

Cutinases are esterases of industrial importance for applications in recycling and surface modification of polyesters. The cutinase from Thielavia terrestris (TtC) is distinct in terms of its ability to retain its stability and activity in acidic pH. Stability and activity in acidic pHs are desirable for esterases as the pH of the reaction tends to go down with the generation of acid. The pH stability and activity are governed by the charged state of the residues involved in catalysis or in substrate binding. In this study, we performed the detailed structural and biochemical characterization of TtC coupled with surface charge analysis to understand its acidic tolerance. The stability of TtC in acidic pH was rationalized by evaluating the contribution of charge interactions to the Gibbs free energy of unfolding at varying pHs. The activity of TtC was found to be limited by substrate binding affinity, which is a function of the surface charge. Additionally, the presence of glycosylation affects the biochemical characteristics of TtC owing to steric interactions with residues involved in substrate binding.

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Acknowledgments

This work was supported by the National Science Foundation’s Chemical, Bioengineering, Environmental and Transport Systems (CBET) Division (Award # 1067415) to R.A.G. and NIH grant R01 GM099827 to C.B. We thank DNA2.0 for gene synthesis and helpful suggestions during the course of this work.

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Authors and Affiliations

  1. Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, CBIS 4105, 110 8th Street, Troy, NY, 12180, USA

    Abhijit N. Shirke, An Su, George Makhatadze & Richard A. Gross

  2. Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, USA

    Abhijit N. Shirke, Danielle Basore, Samantha Holton, An Su, George Makhatadze, Christopher Bystroff & Richard A. Gross

  3. Department of Biological Sciences, Rensselaer Polytechnic Institute, J-Rowl, 3C07, 110 8th Street, Troy, NY, 12180, USA

    Danielle Basore, George Makhatadze, Christopher Bystroff & Richard A. Gross

  4. Department of Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, NY, USA

    Samantha Holton

  5. Center for Genomics and Systems Biology, New York University, New York, NY, USA

    Evan Baugh

  6. Center for Genomics and Systems Biology, New York University Abu Dhabi, Abu Dhabi, United Arab Emirates

    Glenn L. Butterfoss

  7. Department of Computer Science, Rensselaer Polytechnic Institute, Troy, NY, USA

    Christopher Bystroff

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  1. Abhijit N. Shirke
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  2. Danielle Basore
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  4. An Su
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Correspondence to Christopher Bystroff or Richard A. Gross.

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Shirke, A.N., Basore, D., Holton, S. et al. Influence of surface charge, binding site residues and glycosylation on Thielavia terrestris cutinase biochemical characteristics. Appl Microbiol Biotechnol 100, 4435–4446 (2016). https://doi.org/10.1007/s00253-015-7254-1

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