Haloalkane dehalogenases catalyze the hydrolytic cleavage of carbon-halogen bonds, which is a key step in the aerobic mineralization of many environmental pollutants. One important pollutant is the toxic and anthropogenic compound 1,2,3-trichloropropane (TCP). Rational design was combined with saturation mutagenesis to obtain the haloalkane dehalogenase variant DhaA31, which displays an increased catalytic activity towards TCP. Here, the 1.31 Å resolution crystal structure of substrate-free DhaA31, the 1.26 Å resolution structure of DhaA31 in complex with TCP and the 1.95 Å resolution structure of wild-type DhaA are reported. Crystals of the enzyme-substrate complex were successfully obtained by adding volatile TCP to the reservoir after crystallization at pH 6.5 and room temperature. Comparison of the substrate-free structure with that of the DhaA31 enzyme-substrate complex reveals that the nucleophilic Asp106 changes its conformation from an inactive to an active state during the catalytic cycle. The positions of three chloride ions found inside the active site of the enzyme indicate a possible pathway for halide release from the active site through the main tunnel. Comparison of the DhaA31 variant with wild-type DhaA revealed that the introduced substitutions reduce the volume and the solvent-accessibility of the active-site pocket.

Faculty of Science University of South Bohemia in Ceske Budejovice Branisovska 31 370 05 Ceske Budejovice Czech Republic

Institute of Biochemistry Center for Structural and Cell Biology in Medicine University of Lübeck Ratzeburger Allee 160 23538 Lübeck Germany

Institute of Complex Systems FFPW and CENAKVA University of South Bohemia in Ceske Budejovice Zamek 136 373 33 Nove Hrady Czech Republic

Loschmidt Laboratories Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment Faculty of Science Masaryk University Brno Kamenice 5 A13 625 00 Brno Czech Republic

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