Haloalkane dehalogenase from unique plant-pathogenic bacterium Agrobacterium tumefaciens C58
Haloalkane dehalogenases (HLDs) are enzymes catalysing the cleavage of the carbon-halogen bonds. These enzymes have been isolated from various bacteria including degrading, symbiotic, extremophilic or pathogenic strains. HLDs are of great interest due to their possible application in biosensing, bioremediation and industrial biocatalysis.
This research is devoted to the characterization and protein engineering of the new HLD designated DatA from plant pathogenic bacterium Agrobacterium tumefaciens C58. The gene datA is located on the Ti plasmid of A. tumefaciens (GenBank: AE009425). The phylogenetic analysis revelaed that DatA belongs to the HLD-II subfamily. Interestingly, DatA carries a tyrosine instead of tryptophan, which is commonly employed by other HLDs as one of its halide-stabilising residues, necessary for the enzymatic reaction. This speciality together with its origin make DatA a suitable target for a more detailed study.
Biochemical characterization of the successfully expressed and purified DatA revealed some specific properties of this enzyme. Secondary structure of DatA resembles the other characterized HLDs possessing alpha/beta-hydrolase fold. Also the melting temperature (48.3 ± 0.2 °C) is comparable with other HLDs. The activity optimum of DatA was observed at 40 °C and pH 9.8. Native polyacrylamide gel elecrophoresis revealed the dimeric structure of DatA. Analysis of the substrate specificity of DatA revealed its high activity towards brominated and iodinated compounds, but poor activity towards the chlorinated compounds. An unusual substrate-dependent kinetic patterns were obtained during the kinetic analysis of DatA. Most importantly, DatA showed an excellent enantioselectivity towards racemic mixtures of chiral brominated alkanes and esters.
The structural and functional role of the unique halide-stabilising residues was probed by the protein engineering. The halide-stabilising tyrosine 109 was substituted by tryptophan, in order to make the composition of the catalytic pentad of DatA similar to other HLDs. The resulting mutant showed increased melting temperature due to improved hydrophobic packing and strengthened van der Waals contacts in the protein core. The DatA mutant also displayed increased halide binding affinity, but reduced catalytic rate towards most of the substrates. Mutation also modified the kinetic cooperativity and the substrate inhibition, without altering the rate-limiting step of DatA. Biochemically characterized DatA will become a useful biocatalyst in the toolbox of HLDs.