Biochemical Characterization of DpcA from Psychrobacter cryohalolentis K5: The First Extremophilic Member of the Haloalkane Dehalogenase Family

Ivana Drienovská¹, Eva Chovancová¹, Táňa Koudeláková¹, Jiří Damborský¹² and Radka Chaloupková¹

¹Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment, Faculty of Science, Masaryk University, Kamenice 5/A4, 625 00 Brno, ²International Clinical Research Center, St. Anne's University Hospital, 656 91 Brno
drienovska@mail.muni.cz

Haloalkane dehalogenases (HLDs, EC 3.8.1.5) are microbial enzymes which catalyze the hydrolysis of a carbon-halogen bond in halogenated aliphatic hydrocarbons, releasing a halide ion and a corresponding alcohol, as the reaction products. HLDs catalyze the reactions of great environmental and biotechnological significance with potential application in the bioremediation, the biosensing, the decontamination of warfare agents, the synthesis of optically pure compounds, the cellular imaging and the protein tagging. The effective use of biocatalysts in these applications requires availability of enzymes with specific properties under harsh process conditions. Such properties can be easily obtained by isolation of novel biocatalysts from the extremophiles, producing the enzymes called extremozymes. These enzymes are able to operate under extreme conditions such as high or low temperature, high or low pH, pressure or high salinity.

This study describes identification, isolation and biochemical characterization of a novel haloalkane dehalogenase DpcA from Psychrobacter cryohalolentis K5, representing the first extremophilic member of the haloalkane dehalogenase family. This enzyme was purified to homogeneity by metaloaffinity chromatography and biochemically characterized. Correct folding of DpcA was verified by the circular dichroism spectroscopy. Substrate specificity of the enzyme was tested towards thirty different halogenated compounds to confirm its dehalogenase activity. DpcA exhibited one of the narrowest substrate specificity profiles from all biochemically characterized HLDs. It was most active towards 1,3-dibromopropane, 1-bromohexane and 1-bromobutane, generally preferred brominated and terminally substituted substrates. Compared to other HLDs, DpcA has the lowest thermostability (T_m = 34.7 ± 0.6 °C), possesses very unique temperature profile with maximal activity at 25 °C and retains more than 45 % of its activity bellow 15 °C. The psychrophilic properties of DpcA make this enzyme promising biocatalyst for various environmental applications, such as biosensing and/or bioremediation of groundwater contaminated by halogenated pollutants.

This work was supported by the European Regional Development Fund (CZ.1.05/2.1.00/01.0001 and CZ.1.05/1.1.00/02.0123), the Grant Agency of the Czech Republic (P202/10/1435 and P207/12/0775) and the Grant Agency of the Czech Academy of Sciences (IAA401630901).