MS09-P09 Structure analysis of haloalkane dehalogenase DbeA ΔCl variant from Bradyrhizobium elkanii USDA94. Tatyana Prudnikova (Institute of Organic Chemistry and Biochemistry, South Bohemia University , Ceske Budejovice, Czech Republic) Pavlina Rezacova (Institute of Organic Chemistry and Biochemistry and Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic) Radka Chaloupkova (Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment, Faculty of Science, Masaryk University, Brno, Czech Republic) Jiri Damborsky (Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment, Faculty of Science, Masaryk University, Brno, Czech Republic) Ivana Kuta Smatanova (Faculty of Science, University of South Bohemia , Ceske Budejovice, Czech Republic)email: talianensis@gmail.comA novel enzyme, DbeA, belonging to the family of haloalkane dehalogenases (EC 3.8.1.5) was isolated from Bradyrhizobium elkanii USDA94. This haloalkane dehalogenase is closely related to DbjA enzyme from Bradyrhizobium japonicum USDA110 (71% sequence identity), but has different biochemical properties. DbeA is generally less active and has a higher specificity towards brominated and iodinated compounds than DbjA. The DbeA protein was crystallised using the sitting-drop vapour-diffusion method and the crystal structure of a DbeA enzyme has been solved and deposited at Worldwide Protein Data Bank under PDB ID 4k2a. The DbeA wt structure revealed the presence of two halide-binding sites. The first chloride-binding site is located in the active site in between two halide-stabilizing residues. The second halide-binding site is unique to DbeA and has not been previously reported in any other structure of this enzyme family. To elucidate the role of the second halide-binding site, a two-point variant DbeA ΔCl (I44L+Q102H) lacking this site was constructed and biochemically characterized [1]. Elimination of the second halide-binding site decreased the stability and catalytic activity, and dramatically altered the substrate specificity. The two-point substitution resulted in a shift of the substrate-specificity class, which is the first time this has been demonstrated for this enzyme family. Rational design of buried halide-binding sites represents a novel strategy for engineering of enzymes with modified catalytic properties.

The work was supported by the Grant Agency of the Czech Republic P207/12/0775


 
References:

1. Chaloupkova R, et al., Acta Crystallogr. D70, 1884-1897 (2014)
Keywords: Haloalkane dehalogenase, second halide binding site and halogenated compounds