Effect of organic solvents on structure and function of haloalkane dehalogenases

 

Veronika Štěpánková1, Radka Chaloupková1, Jan Brezovský1, Morteza Khabiri2, Babak Minofar1, Zbyněk Prokop1, Rüdiger Ettrich2, and Jiří Damborský1

 

1Loschmidt Laboratories, Institute of Experimental Biology, Masaryk University, Kamenice 5A/4, 625 00 Brno, 2Institute of System Biology and Ecology, Academy of Science of the Czech Republic, Zámek 136, 373 33 Nové Hrady

veronika@chemi.muni.cz, khabiri@greentech.cz

 

The enzymatic catalysis in pure organic solvents or their mixtures with water has developed into active area of research. The utilization of enzymes as highly selective biocatalysts in organic media is being widely applied in the synthesis of many enantiopure compounds [1]. Biochemical properties of enzymes, such as activity, specificity and stability, have been shown to be dependent on the choice of reaction medium [2]. However, not much is known about molecular mechanisms governing the effect of organic solvents on enzymes and their biochemical properties.

The aim of this project is to elucidate the behaviour of three haloalkane dehalogenases (LinB from Sphingobium japonicum UT26, DhaA from Rhodococcus rhodochrous NCIMB13064 and DbjA from Bradyrhizobium japonicum USDA110) in nonconventional media to optimize the conditions for their utilization in industrial applications and to develop enzymes that are catalytically active in the presence of organic solvents. Using kinetic experiments in the presence of various concentrations of thirteen organic solvents we demonstrate, that the tolerance of three studied enzymes to various organic solvents is different even though they belong to the same protein family. DhaA and DbjA are active in aqueous solution as well as in the presence of most of the organic solvents. Moreover, the selected organic solvents have even stimulatory effect on DbjA activity. On the contrary, activity of LinB was significantly decreased in most of the tested solvents. The connection between loss of activity and conformation changes, studied using circular dichroism and fluorescence spectroscopy, was found with DhaA and DbjA, while LinB structure remain mostly intact. To understand the mechanism of interaction of organic solvents with enzymes at the atomic level, the behaviour of LinB, DhaA and DbjA in water and three representative solvents was investigated using molecular dynamics simulations. It was found, that molecules of inhibiting solvents enter the active site in larger number and form a solvent network, while activating solvents are present in active site as a single isolated molecule. The access of solvent molecules to the active site seems to be determined by physico-chemical properties of walls of the tunnel and/or the active site. Kinetic inhibition analysis of enzymes in organic solvents is currently on-going in our laboratory to experimentally verify observations from modelling.

 

This work was financially supported by grant 203/08/0114 of the Grant Agency of Czech Republic, grant IAA401630901 of the Grant Agency of the Academy of Sciences and grants LC06010 and MSM0021622412 of the Ministry of Education.

 

1.     A.M. Klibanov, Nature 409 (2001) 241-246

2.     P. Berglund, Biomol. Eng. 18 (2001) 13-22