Crystallization and preliminary X-ray analysis of DhaA wild type and DhaA13 proteins from Rhodococcus rhodochrous


A. Stsiapanava1, R. Chaloupkova2, A. Jesenska2, J. Brynda3, J. Damborsky2 and

I. Kuta Smatanova1,4


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

2Loschmidt Laboratories, Institute of Experimental Biology, Faculty of Science, Masaryk University, Kamenice 5/A4, 62500 Brno, Czech Republic

3Institute of Molecular Genetics, Academy of Science of the Czech Republic, Flemingovo nam. 2, CZ-16637 Prague 6, Czech Republic

4Institute of Systems Biology and Ecology Academy of Science of the Czech Republic, Zamek 136, 373 33 Nove Hrady, Czech Republic

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Haloalkane dehalogenase DhaA from Rhodococcus rhodochrous NCIMB 13064 is a bacterial enzyme   showing catalytic activity for the hydrolytic conversion of highly toxic industrial pollutant 1,2,3-trichloropropane (TCP) [1,2]. DhaA13 protein variant, carrying mutation H272F in the catalytic histidine, was prepared to catch the protein in a complex with alkyl-enzyme intermediate. This mutant variant binds substrate to the active site, catalyses the first reaction step leading to the formation of the alkyl-enzyme intermediate, but it is not able to convert it further to the product. The main goal of this project is to solve and compare structures of DhaA13 and DhaAwt proteins with two different ligands; environmental pollutant TCP and fluorescence probe coumarine.

DhaAwt and DhaA13 were crystallized using the sitting-drop vapor-diffusion technique [3]. Crystals of DhaAwt grew in the crystallization solution containing 6 % isopropanol and were measured at home diffractometer (IMG, Prague). These crystals diffracted to 1.7 Å. Other crystals of DhaAwt were grown in the solution containing 11 % isopropanol.  These crystals and crystals of DhaA13 soaked in the solution with coumarine were used for synchrotron diffraction measurements at the beamline X12 (EMBL/DESY, Hamburg). Diffracted data for both DhaAwt and DhaA13 crystals were collected to the resolutions limit of 1.26 Ǻ and 1.33 Å, respectively. Protein crystals of DhaAwt obtained in the solution containing TCP and crystals of DhaA13 soaked in the solution of TCP diffracted to ultrahigh resolutions of 1.04 Ǻ and 0.97 Ǻ, respectively. This diffraction data were collected at the beamline 14.1 (BESSY, Berlin). All structures were solved by molecular replacement in monoclinic space group P1. The refinement for DhaAwt and DhaA13 mutant models is currently in progress.


1. T. Bosma, E. Kruizinga, E. J. D. Bruin, G. J. Poelarends & D. B. Janssen, Appl. Environ. Microbiol. 65, (1999),    4575–4581.

2. J. F. Schindler, P. A. Naranjo, D. A. Honaberger, C.-H., Chang,  J. R. Brainard, L. A. Vanderberg & C. J. Unkefer, Biochemistry 38, (1999), 5772–5778.

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


This work is supported by the Ministry of Education of the Czech Republic (MSM6007665808, LC06010) and the Academy of Sciences of the Czech Republic (AV0Z60870520).