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

Haloalkane dehalogenases catalyse environmentally importantdehalogenation reactions. These microbial enzymes representobjects of interest for protein engineering studies, attemptingto improve their catalytic efficiency or broaden their substratespecificity towards environmental pollutants. This paper presentsthe results of a comparative study of haloalkane dehalogenasesoriginating from different organisms. Protein sequences andthe models of tertiary structures of haloalkane dehalogenaseswere compared to investigate the protein fold, reaction mechanismand substrate specificity of these enzymes. Haloalkane dehalogenasescontain the structural motifs of /ß-hydrolases and epoxidaseswithin their sequences. They contain a catalytic triad withtwo different topological arrangements. The presence of a structurallyconserved oxyanion hole suggests the two-step reaction mechanismpreviously described for haloalkane dehalogenase from Xanthobacterautotrophicus GJ10. The differences in substrate specificityof haloalkane dehalogenases originating from different speciesmight be related to the size and geometry of an active siteand its entrance and the efficiency of the transition stateand halide ion stabilization by active site residues. Structurallyconserved motifs identified within the sequences can be usedfor the design of specific primers for the experimental screeningof haloalkane dehalogenases. Those amino acids which were predictedto be functionally important represent possible targets forfuture site-directed mutagenesis experiments.