title: Structural and functional analysis of a novel haloalkane dehalogenase with two halide-binding sites.
abstract: The crystal structure of the novel haloalkane dehalogenase DbeA from Bradyrhizobium elkanii USDA94 revealed the presence of two chloride ions buried in the protein interior. The first halide-binding site is involved in substrate binding and is present in all structurally characterized haloalkane dehalogenases. The second halide-binding site is unique to DbeA. To elucidate the role of the second halide-binding site in enzyme functionality, a two-point mutant lacking this site was constructed and characterized. These substitutions resulted in a shift in the substrate-specificity class and were accompanied by a decrease in enzyme activity, stability and the elimination of substrate inhibition. The changes in enzyme catalytic activity were attributed to deceleration of the rate-limiting hydrolytic step mediated by the lower basicity of the catalytic histidine.
authors.affiliation: Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment RECETOX, Masaryk University, 625 00 Brno, Czech Republic.
authors.forename: Radka
authors.initials: R
authors.lastname: Chaloupkova
authors.affiliation: Faculty of Science, University of South Bohemia in Ceske Budejovice, Branisovska 31, 370 05 Ceske Budejovice, Czech Republic.
authors.forename: Tatyana
authors.initials: T
authors.lastname: Prudnikova
authors.affiliation: Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, 142 20 Prague, Czech Republic.
authors.forename: Pavlina
authors.initials: P
authors.lastname: Rezacova
authors.affiliation: Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment RECETOX, Masaryk University, 625 00 Brno, Czech Republic.
authors.forename: Zbynek
authors.initials: Z
authors.lastname: Prokop
authors.affiliation: Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment RECETOX, Masaryk University, 625 00 Brno, Czech Republic.
authors.forename: Tana
authors.initials: T
authors.lastname: Koudelakova
authors.affiliation: Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment RECETOX, Masaryk University, 625 00 Brno, Czech Republic.
authors.forename: Lukas
authors.initials: L
authors.lastname: Daniel
authors.affiliation: Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment RECETOX, Masaryk University, 625 00 Brno, Czech Republic.
authors.forename: Jan
authors.initials: J
authors.lastname: Brezovsky
authors.affiliation: Graduate School of Life Sciences, Tohoku University, Sendai 980-8577, Japan.
authors.forename: Wakako
authors.initials: W
authors.lastname: Ikeda-Ohtsubo
authors.affiliation: Graduate School of Life Sciences, Tohoku University, Sendai 980-8577, Japan.
authors.forename: Yukari
authors.initials: Y
authors.lastname: Sato
authors.affiliation: Faculty of Science, University of South Bohemia in Ceske Budejovice, Branisovska 31, 370 05 Ceske Budejovice, Czech Republic.
authors.forename: Michal
authors.initials: M
authors.lastname: Kuty
authors.affiliation: Graduate School of Life Sciences, Tohoku University, Sendai 980-8577, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Nagata
authors.affiliation: Faculty of Science, University of South Bohemia in Ceske Budejovice, Branisovska 31, 370 05 Ceske Budejovice, Czech Republic.
authors.forename: Ivana
authors.initials: I
authors.lastname: Kuta Smatanova
authors.affiliation: Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment RECETOX, Masaryk University, 625 00 Brno, Czech Republic.
authors.forename: Jiri
authors.initials: J
authors.lastname: Damborsky
mesh_terms: D001665:Binding Sites; D003460:Crystallization; D006219:Halogens / Q000378:metabolism*; D006867:Hydrolases / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D025341:Principal Component Analysis
chemical_list: D006219:Halogens; D006867:Hydrolases; C046137:haloalkane dehalogenase
keywords: catalytic activity; enzyme stability; halide-binding site; haloalkane dehalogenase; substrate specificity