Crystallization and preliminary diffraction analysis of Dha57, DhaA80 and DhaA106 mutants from Rhodococcus rhodochrous NCIMB 13064

M. Plevaka¹, T.Holubeva¹, P. Rezacova^3,4, T. Prudnikova¹, R. Chaloupkova⁵, T. Koudelakova⁵, I. Kuta Smatanova^1,2

¹University of South Bohemia, Faculty of Science, Branišovska 31, CZ-37005 Česke Budejovice, Czech Republic

²Academy of Sciences of the Czech Republic, Institute of Nanobiology and Structural Biology GCRC, Zamek 136, 373 33 Nove Hrady, Czech Republic

³Institute of Molecular Genetics, Academy of Sciences of the Czech Republic v.v.i., Videnska 1083, 142 20 Prague 4, Czech Republic

⁴Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic v.v.i., Flemingovo nam. 2, 166 37 Prague, Czech Republic

⁵Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment, Faculty of Science, Masaryk University, Kamenice 5/A13, 625 00 Brno, Czech Republic

Haloalkane dehalogenases catalyze a reaction of great environmental and biotechnological significance: conversion of halogenated aliphatic hydrocarbons to their corresponding alcohols. Mutagenesis, focused on the entrance of tunnels in DhaA structure, produced protein variants with significantly improved activity towards 1,2,3-trichloropropane (TCP). The DhaA57, DhaA80 and DhaA106 proteins were constructed in order to determine the 3D structure of the mutants at atomic resolution to explore the importance of tunnel’s mutation to the enzymatic activity to TCP degradation.

The DhaA mutant forms were crystallized using the sitting-drop vapor-diffusion method. Diffraction data for DhaA57 and DhaA80 proteins were collected at the MX14.2 beamline operated by the Helmholtz-Zentrum Berlin (HZB) at the BESSY II electron storage ring (Berlin-Adlershof, Germany), data sets for DhaA106 were collected at the X-ray diffraction station at the X-ray diffraction station at the Institute of Molecular Genetics AS CR in Prague. Diffraction data sets for DhaA57, DhaA80 and DhaA106 proteins were gathered to the 1.20 Å, 1.45 Å and 1.69 Å resolutions, respectively.

The DhaA57 and DhaA80 crystal structures were solved characterized and deposited in RCSB protein data bank under 4F5Z pdb code for DhaA57 and 4F60 pdb code for DhaA80 protein. Model building and refinement of the DhaA106 protein is currently in progress.

This work was supported by the Grant Agency of the Czech Republic (P207/12/0775).