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Balancing the Stability–Activity Trade-Off by Fine-Tuning Dehalogenase Access Tunnels

Authors

  • Veronika Liskova,

    1. Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment RECETOX, Faculty of Science, Masaryk University, Kamenice 5/A13, 625 00 Brno (Czech Republic)
    2. International Clinical Research Center, St. Anne's University Hospital, Pekarska 53, 656 91 Brno (Czech Republic)
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  • David Bednar,

    1. Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment RECETOX, Faculty of Science, Masaryk University, Kamenice 5/A13, 625 00 Brno (Czech Republic)
    2. International Clinical Research Center, St. Anne's University Hospital, Pekarska 53, 656 91 Brno (Czech Republic)
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  • Dr. Tatyana Prudnikova,

    1. Faculty of Science, University of South Bohemia in Ceske Budejovice, Branisovska 31, 370 05 Ceske Budejovice (Czech Republic)
    2. Institute of Nanobiology and Structural Biology, Academy of Sciences of the Czech Republic, Zamek 136, 373 33 Nove Hrady (Czech Republic)
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  • Dr. Pavlina Rezacova,

    1. Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Videnska 1083, 142 20 Prague 4 (Czech Republic)
    2. Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo nam. 2, 166 10 Prague 6 (Czech Republic)
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  • Dr. Tana Koudelakova,

    1. Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment RECETOX, Faculty of Science, Masaryk University, Kamenice 5/A13, 625 00 Brno (Czech Republic)
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  • Dr. Eva Sebestova,

    1. Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment RECETOX, Faculty of Science, Masaryk University, Kamenice 5/A13, 625 00 Brno (Czech Republic)
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  • Assoc. Prof. Ivana Kuta Smatanova,

    1. Faculty of Science, University of South Bohemia in Ceske Budejovice, Branisovska 31, 370 05 Ceske Budejovice (Czech Republic)
    2. Institute of Nanobiology and Structural Biology, Academy of Sciences of the Czech Republic, Zamek 136, 373 33 Nove Hrady (Czech Republic)
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  • Dr. Jan Brezovsky,

    1. Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment RECETOX, Faculty of Science, Masaryk University, Kamenice 5/A13, 625 00 Brno (Czech Republic)
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  • Dr. Radka Chaloupkova,

    Corresponding author
    1. Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment RECETOX, Faculty of Science, Masaryk University, Kamenice 5/A13, 625 00 Brno (Czech Republic)
    • Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment RECETOX, Faculty of Science, Masaryk University, Kamenice 5/A13, 625 00 Brno (Czech Republic)

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  • Prof. Jiri Damborsky

    Corresponding author
    1. Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment RECETOX, Faculty of Science, Masaryk University, Kamenice 5/A13, 625 00 Brno (Czech Republic)
    2. International Clinical Research Center, St. Anne's University Hospital, Pekarska 53, 656 91 Brno (Czech Republic)
    • Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment RECETOX, Faculty of Science, Masaryk University, Kamenice 5/A13, 625 00 Brno (Czech Republic)

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Abstract

A variant of the haloalkane dehalogenase DhaA with greatly enhanced stability and tolerance of organic solvents but reduced activity was created by mutating four residues in the access tunnel. To create a stabilised enzyme with superior catalytic activity, two of the four originally modified residues were randomised. The resulting mutant F 176 G exhibited 32- and 10-times enhanced activity towards 1,2-dibromoethane in buffer and 40 % DMSO, respectively, upon retaining high stability. Structural and molecular dynamics analyses demonstrated that the new variant exhibited superior activity because the F 176 G mutation increased the radius of the tunnel’s mouth and the mobility of α-helices lining the tunnel. The new variant’s tunnel was open in 48 % of trajectories, compared to 58 % for the wild-type, but only 0.02 % for the original four-point variant. Delicate balance between activity and stability of enzymes can be manipulated by fine-tuning the diameter and dynamics of their access tunnels.

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