Crystallization and preliminary crystallographic analysis of Rhodococcus rhodochrous wild-type DhaA protein and its variant DhaA13 complexed with different ligands

 

A. Stsiapanava1, R. Chaloupkova2, A. Fortova2, J. Brynda3, M. S. Weiss4, J. Damborsky2 and I. Kuta Smatanova1,5

 

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

4Helmholtz-Zentrum Berlin, Macromolecular Crystallography (BESSY-MX), Albert-Einstein-Str. 15, D-12489 Berlin, Germany,

5Institute of Systems Biology and Ecology Academy of Science of the Czech Republic, Zamek 136, 373 33 Nove Hrady, Czech Republic

 

The haloalkane dehalogenase DhaA from Rhodococcus rhodochrous NCIMB 13064 can slowly detoxify the industrial pollutant 1,2,3-trichloropropane (TCP) [1,2]. Structural analysis of this enzyme complexed with TCP was conducted to obtain detailed information about the structural limitations of its catalytic properties. The wild-type DhaA was also complexed with two different concentrations of 2-propanol to investigate ability of this ligand to access into the enzyme active site and the effect on enzyme structure. The variant DhaA13 was constructed to terminate the catalytic cycle of enzyme at the stage of the alkyl-enzyme intermediate. The complex with coumarin dye, in which the dye is specifically located in the tunnel mouth of the DhaA13, was used in time-resolved fluorescence spectroscopy to monitor hydration, accessibility and mobility of the dye and its microenvironment in the protein [3]. In our work we aimed to obtain the structures of DhaA13 in complex with coumarin dye and TCP.

All crystallization experiments were performed using the sitting-drop vapour-diffusion method. Diffraction data for wild-type DhaA crystal grown from the solution containing 6% (v/v) 2-propanol was measured at a home source (Institute of Molecular Genetics, Prague). This crystal diffracted to the resolution of 1.75 Å and belonged to the triclinic space group P1. Diffraction data for wild-type DhaA crystal grown from the solution with 11% (v/v) 2-propanol, for DhaA13 crystal soaked with TCP for three hours and for DhaA13 in complex with dye coumarin were collected at the EMBL/DESY in Hamburg. The crystals diffracted to a maximal resolution of 1.26 Å, 1.60 Å and 1.33 Å, respectively. The crystals of wild-type DhaA and variant DhaA13 in complex with dye coumarin belonged to the triclinic space group P1 while the crystal of DhaA13 complexed with TCP belonged to the orthorhombic space group P212121. Data collections for wild-type DhaA crystal grown in the presence of TCP in the crystallization solution and for DhaA13 crystal soaked with TCP for 40 hours were carried at BESSY II in Berlin. The crystals diffracted to maximal resolutions of 1.04 Å and 0.97 Å, respectively. Both crystals belonged to the triclinic space group P1. The structures of wild-type DhaA and variant DhaA13 were solved by molecular replacement using the coordinates from R. rhodochrous haloalkane dehalogenase (PDB code 3FBW) as a search model. Structure refinement of wild-type DhaA and DhaA13 proteins and their complexes are currently in the progress.

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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).