STRUCTURAL CHARACTERIZATION OF THREE DHAA MUTANTS FROM RHODOCOCCUS RHODOCHROUS

A. Stsiapanava¹, J. Dohnalek³, M. Kuty^1,2, J. A. Gavira⁴, T. Koudelakova⁵,

J. 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 the corresponding alcohols [1]. 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.

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 1.22 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. DhaA14 and DhaA15 proteins contain isopropanol in the active site cavity. Mutations in Dha04 and DhaA15 partially block the main tunnel and almost completely block small slot in DhaA14 and DhaA15 enzymes.

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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.