Crystallization and Crystallographic Analysis of a <i>Bradyrhizobium Elkanii</i> USDA94 Haloalkane Dehalogenase Variant with an Eliminated Halide-Binding Site
Tatyana Prudnikova,
Barbora Kascakova,
Jeroen R. Mesters,
Pavel Grinkevich,
Petra Havlickova,
Andrii Mazur,
Anastasiia Shaposhnikova,
Radka Chaloupkova,
Jiri Damborsky,
Michal Kuty,
Ivana Kuta Smatanova
Affiliations
Tatyana Prudnikova
Faculty of Science, University of South Bohemia in Ceske Budejovice, Branisovska 1760, 37005 Ceske Budejovice, Czech Republic
Barbora Kascakova
Faculty of Science, University of South Bohemia in Ceske Budejovice, Branisovska 1760, 37005 Ceske Budejovice, Czech Republic
Jeroen R. Mesters
Institute of Biochemistry, University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany
Pavel Grinkevich
Faculty of Science, University of South Bohemia in Ceske Budejovice, Branisovska 1760, 37005 Ceske Budejovice, Czech Republic
Petra Havlickova
Faculty of Science, University of South Bohemia in Ceske Budejovice, Branisovska 1760, 37005 Ceske Budejovice, Czech Republic
Andrii Mazur
Faculty of Science, University of South Bohemia in Ceske Budejovice, Branisovska 1760, 37005 Ceske Budejovice, Czech Republic
Anastasiia Shaposhnikova
Faculty of Science, University of South Bohemia in Ceske Budejovice, Branisovska 1760, 37005 Ceske Budejovice, Czech Republic
Radka Chaloupkova
Loschmidt Laboratories, Department of Experimental Biology and RECETOX, Faculty of Science, Masaryk University, Kamenice 5/A4, 62500 Brno, Czech Republic
Jiri Damborsky
Loschmidt Laboratories, Department of Experimental Biology and RECETOX, Faculty of Science, Masaryk University, Kamenice 5/A4, 62500 Brno, Czech Republic
Michal Kuty
Faculty of Science, University of South Bohemia in Ceske Budejovice, Branisovska 1760, 37005 Ceske Budejovice, Czech Republic
Ivana Kuta Smatanova
Faculty of Science, University of South Bohemia in Ceske Budejovice, Branisovska 1760, 37005 Ceske Budejovice, Czech Republic
Haloalkane dehalogenases are a very important class of microbial enzymes for environmental detoxification of halogenated pollutants, for biocatalysis, biosensing and molecular tagging. The double mutant (Ile44Leu + Gln102His) of the haloalkane dehalogenase DbeA from Bradyrhizobium elkanii USDA94 (DbeAΔCl) was constructed to study the role of the second halide-binding site previously discovered in the wild-type structure. The variant is less active, less stable in the presence of chloride ions and exhibits significantly altered substrate specificity when compared with the DbeAwt. DbeAΔCl was crystallized using the sitting-drop vapour-diffusion procedure with further optimization by the random microseeding technique. The crystal structure of the DbeAΔCl has been determined and refined to the 1.4 Å resolution. The DbeAΔCl crystals belong to monoclinic space group C121. The DbeAΔCl molecular structure was characterized and compared with five known haloalkane dehalogenases selected from the Protein Data Bank.