STUDY OF WATER MOLECULES DYNAMICS AT THE TUNNEL OPENINGS OF HALOALKANE DEHALOGENASES

Andrea Fořtová¹, Jan Sýkora², Agnieszka Olzyńska², Jan Brezovský¹, Zbyněk Zdráhal³, Martin Hof ² and Jiří Damborský¹

¹Loschmidt Laboratories, Institute of Experimental Biology and National Centre for Biomolecular Research, Faculty of Science, Masaryk University, 625 00 Brno

²J. Heyrovský Institute of Physical Chemistry of the ASCR, 182 23 Prague 8

³Department of Functional Genomics and Proteomics, Institute of Experimental Biology, Faculty of Science, Masaryk University, 625 00 Brno

Haloalkane dehalogenases (3.8.1.5.) are bacterial enzymes cleaving the carbon-halogen bond of halogenated aliphatic compounds by hydrolysis. Active site of haloalkane dehalogenases is buried inside the protein. The active site cavity is connected with the protein surface by tunnels which serve as the transport routes for substrates and products. The tunnel openings belong to the the evolutionally most variable regions among haloalkane dehalogenases. Dynamics of solvent molecules may influence substrate binding and catalytic activity and it is therefore of importance to study solvation dynamics in different proteins.

In this study we employed time resolved fluorescence spectroscopy and molecular dynamic simulations to investigate behavior of water molecules in the vicinity of the tunnel mouth of haloalkane dehalogenases DbjA and DhaA. Specific labeling of DbjA and DhaA is based on formation of a covalent bond between specific ligand and protein during enzymatic reaction. In the wild-type enzyme, this complex is further hydrolyzed by a water molecule which is activated by the catalytic histidine. Histidine substitution impairs hydrolysis step leading to the formation of stable protein ligand complex [1]. In this study, we have developed a protocol for specific labeling of the tunnel opening and elimination of all unbound and non-specifically bound coumarin molecules.

Acrylamide quenching and time-resolved anisotropy experiments confirmed the selective labeling of enzyme by coumarin and complete removal of unbound molecules of coumarin. Steady-state and time-resolved emission spectra measurements showed significant differences in the polarity, accessibility and mobility of the dye and its microenvironment for both studied haloalkane dehalogenases. Coumarin bound in haloalkane dehalogenase DbjA is more flexible and more hydrated in comparison with coumarin bound in DhaA. Microenvironment displays higher polarity and lower viscosity than in DhaA. The obtained experimental data showed good agreement with the results obtained by molecular dynamics calculations. These results reflect geometry of the tunnel mouths evident from the crystal structures [2].

Solvent dynamics in the tunnel mouth will be further studied in other natural haloalkane dehalogenases and their variants. Comparison of solvent dynamics for various constructs will help us to better understand how this dynamics influences functional properties of the enzymes with buried active sites.

[1] HaloTag^TM Interchangeable Labeling Technology, Technical Manual, Promega Corporation, Madison, USA; 2006.

[2] A. Jesenska, J. Sykora, A. Olzynska, J. Brezovsky, Z . Zdrahal, J. Damborsky & M. Hof, J. Am. Chem. Soc. (in press).