Crystallization and preliminary structure analysis of several DhaA mutants from Rhodococcus rhodochrous

A. Stsiapanava¹, J. Dohnalek³, M. Kuty^1,2, Jose A. Gavira⁴, Tana Koudelakova⁵, Jiri Damborsky⁵ and I. Kuta Smatanova^1,2

¹Institute of Physical Biology University of South Bohemia Ceske Budejovice, Zamek 136, 373 33 Nove Hrady, Czech Republic

²Institute of Systems Biology and Ecology Academy of Science of the Czech Republic, Zamek 136, 373 33 Nove Hrady, Czech Republic

³Institute of Macromolecular Chemistry AS CR, Heyrovskeho nam.2, 162 00, Prague 6, Czech Republic

⁴Laboratorio de Estudios Cristalográficos, IACT (CSIC-U. Granada), P.T. Ciencias de la Salud, Granada-18100, Spain

⁵Loschmidt Laboratories, Faculty of Science, Masaryk University, Kamenice 5/A4, 62500 Brno, Czech Republic

stepanova@greentech.cz

Keywords: haloalkane dehalogenases DhaA mutants, X-ray crystallography

Introduction:

Haloalkane dehalogenases (EC 3.8.1.5) are members of the α/β-hydrolase fold family and catalyze hydrolytic conversion of a broad spectrum of hydrocarbons to corresponding alcohols [1]. These enzymes are potentially important biocatalysts for industrial and bioremediation applications.
Besides a wide range of haloalkanes, DhaA can slowly convert serious industrial pollutant 1,2,3-trichloropropane (TCP) [2]. Three mutants marked as DhaA04, DhaA14 and DhaA15 were designed and constructed to study the relevance of the tunnels connecting the buried active site with the surrounding solvent for the enzymatic activity.

Results and discussion:

The three mutants of DhaA were crystallized using the sitting-drop vapor-diffusion technique [3]. Crystal growth conditions were optimized [4] and crystals were used for synchrotron diffraction measurements at the beamline X11 of the DORIS storage ring at the EMBL Hamburg Outstation. X-ray intensities data for DhaA04, DhaA14 and DhaA15 mutants were collected to a resolutions limit of 1.23 Ǻ, 0.95 Å and 1.15 Å, respectively. Crystals of DhaA04 belong to the orthorhombic space group P2₁2₁2₁ while crystals of DhaA14 and DhaA15 mutants belong to the triclinic space group P1. The known structure of the haloalkane dehalogenase from Rhodococcus species (PDB code 1bn6) [5] was used as a template for the molecular replacement. Analyses of crystal structures of mutants allow determine of electron densities observed for the ligands. In the case of DhaA04 the ligand is benzoic acid. DhaA15 protein contains isopropanol in the active site cavity. The refinement for DhaA14 mutant model and the initial interpretation of the structural implication in protein activities is currently in progress.

References:

1. D. B. Janssen, Curr. Opin. Chem.l Biol., 8, (2004), 150‑159.

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).

4. A. Stsiapanava, T. Koudelakova, M. Lapkouski, M. Pavlova, J. Damborsky & I. Kuta Smatanova, Acta Cryst. F64, (2008), 137-140.

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

Acknowledgement:

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).