Atomic resolution studies of haloalkane dehalogenases DhaA04, DhaA14 and DhaA15 with engineered access tunnels.

The haloalkane dehalogenase DhaA from Rhodococcus rhodochrous NCIMB 13064 is a bacterial enzyme that shows catalytic activity for the hydrolytic degradation of the highly toxic industrial pollutant 1,2,3-trichloropropane (TCP). Mutagenesis focused on the access tunnels of DhaA produced protein variants with significantly improved activity towards TCP. Three mutants of DhaA named DhaA04 (C176Y), DhaA14 (I135F) and DhaA15 (C176Y + I135F) were constructed in order to study the functional relevance of the tunnels connecting the buried active site of the protein with the surrounding solvent. All three protein variants were crystallized using the sitting-drop vapour-diffusion technique. The crystals of DhaA04 belonged to the orthorhombic space group P2(1)2(1)2(1), while the crystals of DhaA14 and DhaA15 had triclinic symmetry in space group P1. The crystal structures of DhaA04, DhaA14 and DhaA15 with ligands present in the active site were solved and refined using diffraction data to 1.23, 0.95 and 1.22 A, resolution, respectively. Structural comparisons of the wild type and the three mutants suggest that the tunnels play a key role in the processes of ligand exchange between the buried active site and the surrounding solvent.

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.