September 15-19, 2019
Improvements in the catalytic activity, substrate specificity or enantioselectivity of enzymes are traditionally achieved by modification of enzymes’ active sites. We have recently proposed that the enzyme engineering endeavors should target both the active sites and the access tunnels/channels [1,2]. Using the model enzymes haloalkane dehalogenases, we have demonstrated that engineering of access tunnels provides enzymes with significantly improved catalytic properties  and stability . User-friendly software tools Caver , Caver Analyst , CaverDock  and Caver Web , have been developed for the computational design of protein tunnels/channels; FireProt  and HotSpot Wizard  for automated design of stabilizing mutations and smart libraries. Using these tools we were able to introduce a new tunnel to a protein structure and tweak its conformational dynamics. This engineering strategy has led to improved catalytic efficiency , enhanced promiscuity or even a functional switch (unpublished). Our concepts and software tools are widely applicable to various enzymes with known structures and buried active sites.
1. Damborsky, J., et al., 2009: Computational Tools for Designing and Engineering Biocatalysts. Current Opinion in Chemical Biology 13: 26-34.
2. Prokop, Z., et al., 2012: Engineering of Protein Tunnels: Keyhole-lock-key Model for Catalysis by the Enzymes with Buried Active Sites. Protein Engineering Handbook, Wiley-VCH, Weinheim, pp. 421-464.
3. Brezovsky, J., et al., 2016: Engineering a de Novo Transport Tunnel. ACS Catalysis 6: 7597-7610.
4. Koudelakova, T., et al., 2013: Engineering Enzyme Stability and Resistance to an Organic Cosolvent by Modification of Residues in the Access Tunnel. Angewandte Chemie 52: 1959-1963.
5. Chovancova, E., et al., 2012: CAVER 3.0: A Tool for Analysis of Transport Pathways in Dynamic Protein Structures. PLOS Computational Biology 8: e1002708.
6. Jurcik, A., et al., 2018: CAVER Analyst 2.0: Analysis and Visualization of Channels and Tunnels in Protein Structures and Molecular Dynamics Trajectories. Bioinformatics 34: 3586-3588.
7. Vavra, O., et al., 2019: CaverDock 1.0: A New Tool for Analysis of Ligand Binding and Unbinding Based on Molecular Docking. Bioinformatics (under review).
8. Stourac, J., et al. 2019: Caver Web 1.0: Identification of Tunnels and Channels in Proteins and Analysis of Ligand Transport. Nucleic Acids Research (under review).
9. Musil, M., et al., 2017: FireProt: Web Server for Automated Design of Thermostable Proteins. Nucleic Acids Research 45: W393-W399.
10. Sumbalova, L. et al., 2018: HotSpot Wizard 3.0: Automated Design of Site-Specific Mutations and Smart Libraries in Protein Engineering. Nucleic Acids Research 46: W356-W362.
Jiri Damborsky, David Bednar, Sergio Marques, Piia Kokkonen, Gaspar Pinto, Joan Planas Iglesias, Milos Musil, Jiri Hon, Lenka Sumbalova, Ondrej Vavra, Jiri Filipovic, Bara Kozlikova, Martin Marek, Zbynek Prokop, and Jan Stourac, "Strategies and software tools for engineering protein tunnels and dynamical gates" in "Enzyme Engineering XXV", Huimin Zhao, University of Illinois at Urbana-Champaign, USA John Wong, Pfizer, USA Eds, ECI Symposium Series, (2019). https://dc.engconfintl.org/enzyme_xxv/138