We emphasize the importance of dynamics and hydration for enzymatic catalysis and protein design by transplanting the active site from a haloalkane dehalogenase with high enantioselectivity to nonselective dehalogenase. Protein crystallography confirms that the active site geometry of the redesigned dehalogenase matches that of the target, but its enantioselectivity remains low. Time-dependent fluorescence shifts and computer simulations revealed that dynamics and hydration at the tunnel mouth differ substantially between the redesigned and target dehalogenase.

] Faculty of Science University of South Bohemia in Ceske Budejovice Ceske Budejovice Czech Republic [2] Institute of Nanobiology and Structural Biology ASCR v v i Nove Hrady Czech Republic

] J Heyrovsky Institute of Physical Chemistry of the ASCR v v i Academy of Sciences of the Czech Republic Prague Czech Republic [2]

] Loschmidt Laboratories Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment Faculty of Science Masaryk University Brno Czech Republic [2]

] Loschmidt Laboratories Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment Faculty of Science Masaryk University Brno Czech Republic [2] International Clinical Research Center St Anne's University Hospital Brno Brno Czech Republic

J Heyrovsky Institute of Physical Chemistry of the ASCR v v i Academy of Sciences of the Czech Republic Prague Czech Republic

Loschmidt Laboratories Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment Faculty of Science Masaryk University Brno Czech Republic

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