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
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 . Crystals of DhaAwt grew in the
crystallization solution containing 6 % isopropanol and were measured at home diffractometer
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).
work is supported by the Ministry of Education of the