Haloalkane dehalogenases (HLDs) are microbial enzymes that catalyze the cleavage of a carbon-halogen bond by a hydrolytic mechanism. The reaction products are halide ion, hydronium ion (proton) and an alcohol molecule. These enzymes can be useful for the biodegradation of many important environmental pollutants such as 1,2-dichloroethane, 1,2,3,4,5,6-hexachlorocyclohexane, and 1-chlorobutane [1].
The haloalkane dehalogenase DhaA from Rhodococcus rhodochrous is a monomeric
enzyme with molecular weight of 34 kDa and it is a representative of the large
superfamily of α/β‐hydrolases [1].
Mutant variant DhaA80 with enhanced structural and kinetic stability in the
presence of dimethyl sulfoxide and the elevated temperature was constructed by
directed evolution and site-directed mutagenesis. The crystallization
experiment was performed using the sitting-drop vapor-diffusion method at a
temperature of 4°C. Crystals of DhaA80 grown from the precipitant containing
20% PEG 3350, 0.2M sodium fluoride and ionic liquids (IL) from Hampton
Research Ionic Liquid Screen - HR2-214. Crystals diffracted to the
resolution of 1.8Å. The known structure of HLD from Rhodococcus
sp. (www.rcsb.org) was used as a template for molecular replacement. The
structures of DhaA80 including IL solutions containing 1) 50% w/v
2-Hydroxyethylammonium formate, 2) 50% w/v 1-Butyl-3-methylimidazolium
dicyanamide and 3) 50% w/v 1-Butyl-3-methylimidazolium methyl sulfate were
solved after several refinements and validations. For further research and
understanding, the interaction of ILs ions molecular dynamics (MD) simulations have
been used. MD simulations of systems containing DhaA80 with 50% w/v
1-Butyl-3-methylimidazolium methyl sulfate and 50% w/v 2-Hydroxyethylammonium
formate have been performed. The analysis of MD data by using root mean square
deviation (RMSD) and root mean square fluctuations (RMSF) confirmed that ILs
affect the structure and stability of enzyme.