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Protein Engineering, Vol. 12, No. 11, 989-998, November 1999
© 1999 Oxford University Press

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

Jirí Damborsky1 and Jaroslav Koca

Laboratory of Biomolecular Structure and Dynamics, Faculty of Science, Masaryk University, Kotlarska 2, 611 37 Brno, Czech Republic

Haloalkane dehalogenases catalyse environmentally important dehalogenation reactions. These microbial enzymes represent objects of interest for protein 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 dehalogenases originating from different organisms. Protein sequences and the models of tertiary structures of haloalkane dehalogenases were compared to investigate the protein fold, reaction mechanism and substrate specificity of these enzymes. Haloalkane dehalogenases contain the structural motifs of {alpha}/ß-hydrolases and epoxidases within their sequences. They contain a catalytic triad with two different topological arrangements. The presence of a structurally conserved oxyanion hole suggests the two-step reaction mechanism previously described for haloalkane dehalogenase from Xanthobacter autotrophicus GJ10. The differences in substrate specificity of haloalkane dehalogenases originating from different species might be related to the size and geometry of an active site and its entrance and the efficiency of the transition state and halide ion stabilization by active site residues. Structurally conserved motifs identified within the sequences can be used for the design of specific primers for the experimental screening of haloalkane dehalogenases. Those amino acids which were predicted to be functionally important represent possible targets for future site-directed mutagenesis experiments.

Keywords: haloalkane dehalogenase/homology modelling/protein fold/reaction mechanism/substrate specificity

1 To whom correspondence should be addressed


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