Preliminary structure characterization of DHAA mutants from RHODOCOCCUS RHODOCHROUS

A. Stsiapanava¹, J. Dohnalek³, M. Kuty^1,2, M. Lapkouski¹ , 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 Cristalografico, Edificio BIC-Granada, Avda. de la Innovacion 1, P.T. Ciencias de la Salud, 18100-Armilla, Granada, Spain

⁵<aff><oid id="5551Loschmidt Laboratories, Faculty of Science, Masaryk University, Kamenice 5/A4, 62500 Brno, Czech Republic"> </cny></aff></aug>

stepanova@greentech.cz

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 constructed to study importance of tunnels connecting buried active site with the surrounding solvent for the enzymatic activity.

All mutant proteins were crystallized using a sitting-drop vapor-diffusion technique [3]. Grow conditions were optimized [4] and crystals were used for synchrotron diffraction measurements at the beamline X11 of a DORIS storage ring at the EMBL Hamburg Outstation.

Diffraction data for DhaA04, DhaA14 and DhaA15 mutants were collected to the high resolutions 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 second two mutants DhaA14 and DhaA15 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. Currently, structures of the DhaA mutant proteins are in the process of being further refined and interpreted.

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.

The authors thank Jindrich Hasek (Academy of Sciences of the Czech Republic, Prague) and Juan Manuel Garcia-Ruiz (Laboratorio de Estudios Cristalografico, Edificio BIC-Granada) for their generous support. 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). We are grateful to X11 Consortium for Protein Crystallography for access to their facility.