MS04-P04 Structural characterization and comparison of crystallization behaviour of selected haloalkane dehalogenases Ivana Kuta Smatanova (Institute of Chemistry and Biochemistry, Faculty of Science, University of South Bohemia Ceske Budejovice, Ceske Budejovice, Czech Republic) Tatyana Prudnikova (Institute of Chemistry and Biochemistry, Faculty of Science, University of South Bohemia Ceske Budejovice, Ceske Budejovice, Czech Republic) Radka Chaloupkova (Loschmidt Laboratories, Faculty of Science, Masaryk University Brno, Brno, Czech Republic) Michal Kuty (Institute of Chemistry and Biochemistry, Faculty of Science, University of South Bohemia Ceske Budejovice, Ceske Budejovice, Czech Republic) Andrii Mazur (Institute of Chemistry and Biochemistry, Faculty of Science, University of South Bohemia Ceske Budejovice, Ceske Budejovice, Czech Republic) Barbora Kascakova (Institute of Chemistry and Biochemistry, Faculty of Science, University of South Bohemia Ceske Budejovice, Ceske Budejovice, Czech Republic) Jiri Damborsky (Loschmidt Laboratories, Faculty of Science, Masaryk University Brno, Brno, Czech Republic) Pavlina Rezacova (Institute of Organic Chemistry and Biochemistry and Institute of Molecular Genetics AS CR Praha, Praha, Czech Republic)email: ivanaks@seznam.czHalogenated aliphatic compounds represent one of the largest groups of environmental pollutants. Haloalkane dehalogenases are responsible for one of the key reactions in the bacterial degradation of various halogenated pollutants. Apart from applications in bioremediation, haloalkane dehalogenases can be potentially applied in biosensing of pollution, biosynthesis, cellular imaging and protein immobilization. These enzymes catalyze the cleavage of a carbon-halogen bond in haloalkanes with water as the sole co-substrate, resulting in formation of a halide ion, a corresponding alcohol, and a proton. The role of conformational flexibility has been well established in connection with the accessibility of the active site, the binding of substrates and ligands, and release of products, stabilization and trapping of intermediates, orientation of the substrate into the binding cleft or adjustment of the reaction environment.
Several haloalkane dehalogenase structures were determined by X-ray diffraction analysis of enzymes’ crystals, providing a good theoretical framework for their modification by protein engineering. Crystallization conditions for haloalkane dehalogenases DhaA from Rhodococcus rhodochrous NCIMB 13064, LinB from Sphingobium japonicum UT26, and DbeA from Bradyrhizobium elkanii USDA94 and their mutant variants were compared and analyzed. Analysis of crystallization cocktails revealed common components for the majority of compared dehalogenases such as divalent cations such as calcium or magnesium, and medium size polyethylene glycols (PEGs) 3350 or 4000 as well as almost neutral pH. Instead of X-ray diffraction analysis our model systems have been also investigated by other structural methods such as neutron crystallography, time-resolved crystallography and hydrogen-deuterium exchange mass spectrometry. Based on carefully designed experiments and by combination of the information obtained from different, but complementary, techniques we will be able to get inside into (i) conformational changes of selected enzymes upon their interactions with substrates, (ii) location of hydrogen atoms inside the enzyme active site and the access tunnels and (iii) protonation state of catalytic residues of the enzymes during their catalytic cycles.

This research is supported by the GACR (17-24321S).
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Keywords: haloalkane dehalogenases, crystallization, structure