Crystallization and preliminary structure analysis of DhaA57 and DhaA80 mutants from Rhodococcus rhodochrous

 

Maryia Plevaka1, 2, Daryna Kulik1, 2, Radka Chaloupkova3, Tana Koudelakova3, Pavlina Rezacova4, 5, Michal Kuty1, 6, Jiri Damborsky3 and Ivana Kuta Smatanova1, 6

 

1 University of South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses and School of complex systems, Zamek 136, 373 33 Nove Hrady

2 University of South Bohemia in Ceske Budejovice, Faculty of Science, Branisovska 31, 37005 Ceske Budejovice

3 Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment, Faculty of Science, Masaryk University, Kamenice 5/A4, 625 00 Brno, Czech Republic

4 Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Flemingovo nam. 2, 166 37 Prague, Czech Republic

5 Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo nam. 2, 166 37 Prague, Czech Republic

6 Academy of Sciences of the Czech Republic, Inst. of Nanobiology and Structural Biology GCRC, Zamek 136, 373 33 Nove Hrady

 

Haloalkane dehalogenases catalyze a reaction of great environmental and biotechnological significance: conversion of halogenated aliphatic hydrocarbons to their corresponding alcohols [1]. Practical use of these enzymes could be significantly improved by the availability of biocatalysts stable in the presence of organic co-solvents and at elevated temperature. New mutant variants of DhaA from Rhodococcus rhodochrous DhaA57 and DhaA80 with enhanced structural and kinetic stability in the presence of dimethyl sulfoxide and elevated temperature were recently constructed by directed evolution and site-directed mutagenesis. The main goal of presented project is to determine the 3D structure of the DhaA57 and DhaA80 mutants at atomic resolution in order to explore the effects of mutations on the enzymatic activity of modified protein from a structural perspective.

Crystallization experiments were performed using the sitting-drop vapor-diffusion method at temperature of 4°C. Crystals of DhaA57 grown from the precipitant containing 35% PEG 4000; and DhaA80 crystals were appeared in the solution consisting of 20% PEG 3350 and 0.2M sodium fluoride. Both crystals were tested on the home diffractometer at the IOCB & IMG AS CR in Prague. Diffraction data sets for DhaA57 and DhaA80 were collected at the synchrotron EMBL/DESY in Hamburg (Germany). Crystals diffracted to the resolution of 1.8Å for DhaA57 and 1.9Å for DhaA80, respectively. The DhaA57 crystals belong to the primitive triclinic space group P1, while the DhaA80 crystals to the primitive orthorhombic space group P222. The known structure of the haloalkane dehalogenase from Rhodococcus sp. [3] will be used as a template for molecular replacement. The process of the DhaA57 and DhaA80 structure refinement is currently in the progress.

This research was supported by the GACR (P207/12/0775), ME CR (CZ.1.05/2.1.00/01.0024), by the AS CR (AV0Z60870520) and GAJU 170/2010/P for MP and DK.

1.       Janssen, D. B., Dinkla, I. J. T., Poelarends, G. J. & Terpstra, P. (2005). Environ. Microbiol. 7, 1868–1882.

2.       Newman J, Peat T.S., Richard R., Kan L., Swanson P.E., Affholter E.A., Holmes I.H., Schindler J.F., Unkefer J.C. & Terwilliger T.C., Biochemistry, 38, (1999), 16105‑16114.