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 . 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
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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).