Crystallization study of Rhodococcus rhodochrous mutant protein DhaA31

Maryna Sviatlova^a, Monika Strakova^c, Radka Chaloupkova^c_, Jiri Damborsky^c and Ivana Kuta Smatanova^a,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

DhaA31 is haloalkane dehalogenase isolated from bacterium Rhodococcus rhodochrous NCIMB 13064 [1]. Haloalkane dehalogenases are enzymes that catalyze the cleavage of the carbon-halogen bond by a hydrolytic mechanism [2]. These proteins are key enzymes for the degradation of many halogenated aliphatic pollutants [3]. So haloalkane dehalogenases are potentially important biocatalysts with both industrial and bioremediation applications that could be used for industrial biocatalysis and as active compounds of biosensors, respectively [4].

The goal in our project is produce good crystals of DhaA31 for diffraction experiments. Structure study of DhaA31 allows us to determine importance of protein mutations on enzyme functions.

Standard vapor diffusion technique (sitting drop) has been used for searching and optimization of crystallization conditions [5]. In crystallization experiments have been used Hampton Research Linbro and Cryschem plates (Hampton Research, CA, USA), Emerald BioStructures CombiClover Crystallization Plate (EBS plate, Emerald BioStructures, WA, USA). Microcrystals have been obtained by sparse matrix screening using commercial crystallization kits as Crystal Screen of Hampton Research, Emerald BioSystem and Jena BioScience. Crystallization experiments are in the progress.

1. Kulakova, A. N., Larkin, M. J. & Kulakov, L. A. (1997). Microbiology 143, 109–115.

2.. Jesenska, A., Pavlova, M., Strouhal, M., Chaloupkova, R., Tesinska, I., Monincova, M., Prokop, Z., Bartos, M., Pavlik, I., Rychlik, I., Mobius, P., Nagata, Y., Damborsky, J. (2005). Cloning, Biochemical Properties, and Distribution of Mycobacterial Haloalkane Dehalogenases. Appl. Environ. Microbiol. 71: 6736-6745

3. Sato, Y., Monincova, M., Chaloupkova, R., Prokop, Z., Ohtsubo, Y., Minamisawa, K., Tsuda, M., Damborsky, J., Nagata, Y. (2005). Two Rhizobial Strains, Mesorhizobium loti MAFF303099 and Bradyrhizobium japonicum USDA110, Encode Haloalkane Dehalogenases with Novel Structures and Substrate Specificities. Appl. Environ. Microbiol. 71: 4372-4379

4. Stsiapanava A., Koudelakova T., Lapkouski M., Pavlova M., Damborsky J. and Kuta Smatanova I. Crystals of DhaA mutants from Rhodococcus rhodochrous NCIMB 13064 diffracted to ultrahigh resolution: crystallization and preliminary diffraction analysis Acta Cryst. (2008). F64, 137-140

5.Ducruix, A. & Giegé, R. (1999). Crystallization of Nucleic Acids and Proteins: A Practical Approach, 2nd Ed. Oxford: Oxford University Press.

Acknowledgements:

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).