Crystallization study of three mutant haloalkane dehalogenases derived from dehalogenase DhaA of Rhodococcus rhodochrous NCIMB 13064
Alena Stsiapanavaa, Tana Koudelakovac, Lucie Grodeckac, Jiri Damborskyc, and Ivana Kuta Smatanovaa,b
aInstitute of Physical Biology University of South Bohemia Ceske Budejovice, Zamek 136, 373 33 Nove Hrady, Czech Republic
bInstitute of Systems Biology and Ecology Academy of Science of the Czech Republic, Zamek 136, 373 33 Nove Hrady, Czech Republic
cLoschmidt Laboratories, Faculty of Science, Masaryk University, Kamenice 5/A4, 62500 Brno, Czech Republic
Haloalkane dehalogenases (EC 188.8.131.52) are enzymes that belong to the α/β-hydrolase fold family. These microbial enzymes catalyze hydrolytic conversion of halogenated hydrocarbons to corresponding alcohols . Dehalogenation is a key step in aerobic mineralization pathways of many halogenated compounds that occur as environmental pollutants . Haloalkane dehalogenases are potentially important biocatalysts with both industrial and bioremediation applications that could be used for industrial biocatalysis or as active compounds of biosensors, respectively [3, 4].
Wild-type DhaA was isolated from bacterium Rhodococcus rhodochrous NCIMB 13064 . Derived mutant enzymes DhaA04, DhaA14 and DhaA15 were constructed to reveal importance of product transporting pathways (tunnels) in DhaA for its enzymatic activity. Our project is aimed to produce crystals of haloalkane dehalogenases DhaA04, DhaA14 and DhaA15 purified mutants (Fig. 1) in efficient quality for diffraction experiments and finally compare results with known structure of wild-type DhaA .
Standard vapor diffusion technique has been used for searching and optimization of crystallization conditions. Crystallization experiments have been performed in Hampton Research Linbro and Cryschem plates (Hampton Research, CA, USA) as well as in Emerald BioStructures CombiClover Crystallization Plate (EBS plate, Emerald BioStructures, WA, USA) using commercial crystallization kits as Crystal Screen Lite and Crystal Screen of Hampton Research, and Clear Strategy Screen 1 of Molecular Dimensions Limited (MDL, Suffolk, UK). The first microcrystals of DhaA15 were obtained from PCT, reagent B2 of Hampton Research (Fig. 2).
Crystallization experiments with all enzymes are in the progress.
This work is supported by the Ministry of Education of the Czech Republic (MSM6007665808 and LC06010) and by the Academy of Sciences of the Czech Republic (AVOZ60870520).
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