Computational site-directed mutagenesis of haloalkane dehalogenase in position 172

The application of molecular modelling and quantum-chemistry calculations for the 'computational site-directed mutagenesis' of haloalkane dehalogenase is described here. The exhaustive set of single point mutants of haloalkane dehalogenase in position 172 was constructed by homology modelling. The ability of substituting residues to stabilize the halide ion formed during the dehalogenation reaction in the enzyme active site was probed by quantum-chemical calculations. A simplified modelling procedure was adopted to obtain informative results on the potential activity of mutant proteins in a sufficiently short period of time, which, in the future, could be applicable for making bona fide predictions of mutants' activity prior to their preparation in the laboratory. The reaction pathways for the carbon-halide bond cleavage were calculated using microscopic models of wild type and mutant proteins. The theoretical parameters derived from the calculation, i.e. relative energies and selected atomic charges of educt, product and transition state structures, were statistically correlated with experimentally determined activities. The charge difference of educt and product on the halide-stabilizing hydrogen atom of residue 172 was the best parameter to distinguish protein variants with high activity from mutant proteins displaying a low activity. All mutants with significant activity in the experiment were found to have this parameter one order of magnitude higher than mutants with low activity. The results obtained are discussed in the light of the practical application of this methodology for the prediction of potentially active protein variants. Further automation of the modelling procedure is suggested for combinatorial screening of the large number of protein variants. Coupling of the dehalogenation reaction with hydrogenation of the halide ion formed during the reaction in the enzyme active site was proposed as a possible way to improve the catalytic activity of the haloalkane dehalogenase of Xanthobacter autotrophicus GJ10.