Crystallization and structural analysis of DhaA31 protein from Rhodococcus rhodochrous

M. Lahoda¹, R. Chaloupkova², A. Stsiapanava¹, J. Damborsky² and I. Kuta-Smatanova^{1, 3}

¹Institute of Physical Biology, University of South Bohemia, Ceske Budejovice, Zamek 136, 373 33 Nove Hrady, Czech Republic

²Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment, Faculty of Science, Masaryk University, Kamenice 5/A4, 62500 Brno

³Institute of Systems Biology and Ecology, Academy of Science of the Czech Republic, Zamek 136, 373 33 Nove Hrady, Czech Republic

Haloalkane dehalogenase DhaA is a bacterial enzyme isolated from bacterium Rhodococcus rhodochrous NCIMB 13064 [1]. The enzyme catalyzes hydrolytic dehalogenation of various halogenated aliphatic hydrocarbons [2]. The mutant DhaA31 was constructed to degrade anthropogenic compound 1,2,3-trichloropropane (TCP). TCP has been detected in low concentrations in surface water, drinking water and groundwater. TCP shows strict resistant to biological degradation. To increase the efficiency of this reaction, focused directed evolution was used to construct the mutant DhaA31 with up to 32-fold higher catalytic activity and 26-fold higher catalytic efficiency, than parent wild type enzyme DhaA [3]. The main goal of this project is to solve structure of DhaA31. The structure of DhaA31 will help to understand the structure-function relationships of improved dehalogenation of TCP.

The mutant protein DhaA31 was crystallized by sitting drop vapor diffusion technique and crystals of DhaA31 in a complex with TCP were obtained using soaking experiment. Both crystals belong to the triclinic space group P1. Diffraction data were collected to the high resolution of 1.31 Å for DhaA31 and 1.26 Å for DhaA31 with TCP. This diffraction data were collected at the beamline X12 (DESY, Hamburg). The structures of DhaA31 and DhaA31 in a complex with TCP were solved by molecular-replacement techniques using haloalkane dehalogenase DhaA04 mutant as a template. The refinement has been done in SHELXL program [4].

This work is supported by the Ministry of Education of the Czech Republic (LC06010, MSM6007665808, MSM0021622412 and CZ.1.05/2.1.00/01.0001) and the Academy of Sciences of the Czech Republic (AV0Z60870520 and IAA401630901).

1. Kulakova, A. N., Larkin, M. J. & Kulakov, L. A. (1997). Microbiol. 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. Pavlova, M., Klvana, M., Prokop, Z., Chaloupkova, R., Banas, P., Otyepka, M., Wade, R. C., Tsuda, M., Nagata, Y. & Damborsky, J. (2009). Nature Chem. Biol. 5, 727-733.

4. Sheldrick, G.M. (2008). Acta Cryst. A64: 112–122.