Crystallization and crystallography analysis of haloalkane dehalogenase mutant DhaA12 from Rhodococcus rhodochrous
Maryna Lahodaa, Tana Koudelakovab, Jiri Damborskyb and Ivana Kuta Smatanovaa,c
aInstitute of Physical Biology University of South Bohemia Ceske Budejovice, Zamek 136, 373 33 Nove Hrady, Czech Republic
bLoschmidt Laboratories, Faculty of Science, Masaryk University, Kamenice 5/A4, 62500 Brno, Czech Republic
cInstitute of Systems Biology and Ecology Academy of Science of the Czech Republic, Zamek 136, 373 33 Nove Hrady, Czech Republic
Haloalkane dehalogenases comprise a group of enzymes that hydrolyse carbon-halogen bounds in a wide range of haloalkanes . DhaA12 mutant contains several of amino acid residues typical for haloalkane dehalogenase DbjA that were introduce into DhaA. DbjA shows significant enantioselectivity with selected substrates in contrast to DhaA that shows no enantioselectivity . Enantioselectivity plays an important role in cleaning substratum in the production of pharmaceuticals and agrochemicals. The aim of creating the DhaA12 enzyme was to enhance the enantioselectivity of DhaA to the extent of the DbjA enantioselectivity. The goal of this project is solving of protein structure to understand the changes in the enantioselectivity and possibly for further design of mutations.
DhaA12 protein was crystallized using sitting-drop vapor-diffusion technique . Crystals of DhaA12 grew at room temperature in the crystallization solution containing 20% PEG 4000, 100mM MES Sodium salt, pH 6.1. Crystallization experiments have been performed in CombiClover Crystallization Plates (EBS plate, Emerald BioStructures, WA, USA). Diffraction data for DhaA12 was collected at the beamline 14.1 BESSY (Berlin) to the maximum resolution of 1.68 Ǻ. Crystals of mutant belong to the orthorhombic space group P212121. The known structure of the haloalkane dehalogenase from Rhodococcus sp.  was used as a template for molecular replacement.The process of the DhaA12 structure refinement is in progress.
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