MS09-P09 Structure analysis of haloalkane dehalogenase DbeA ΔCl variant from Bradyrhizobium elkanii USDA94.
A novel enzyme, DbeA, belonging to the family of haloalkane dehalogenases (EC 220.127.116.11) 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 . 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
1. Chaloupkova R, et al., Acta Crystallogr. D70, 1884-1897 (2014)Keywords: Haloalkane dehalogenase, second halide binding site and halogenated compounds