Analysis of the reaction mechanism and substrate specificity of haloalkanedehalogenases by sequential and structural comparisons

Haloalkane dehalogenases catalyse environmentally important dehalogenation reactions. These microbial enzymes represent objects of interest for protei n engineering studies, attempting to improve their catalytic efficiency or broaden their substrate specificity towards environmental pollutants. This paper presents the results of a comparative study of haloalkane dehalogenas es originating from different organisms. Protein sequences and the models o f tertiary structures of haloalkane dehalogenases were compared to investig ate the protein fold, reaction mechanism and substrate specificity of these enzymes. Haloalkane dehalogenases contain the structural motifs of alpha/b eta-hydrolases and epoxidases within their sequences. They contain a cataly tic triad with two different topological arrangements. The presence of a st ructurally conserved oxyanion hole suggests the two-step reaction mechanism previously described for haloalkane dehalogenase from Xanthobacter autotro phicus GJ10. The differences in substrate specificity of haloalkane dehalog enases originating from different species might be related to the size and geometry of an active site and its entrance and the efficiency of the trans ition state and halide ion stabilization by active site residues. Structura lly conserved motifs identified within the sequences can be used for the de sign of specific primers for the experimental screening of haloalkane dehal ogenases. Those amino acids which were predicted to be functionally importa nt represent possible targets for future site-directed mutagenesis experime nts.