Molecular mechanisms of activation and inhibition of haloalkane dehalogenases by organic co-solvents

V. Stepankova¹, J. Brezovsky¹, M. Khabiri², A. Pavelka^1,3,4, Z. Prokop¹, R. Ettrich², J. Damborsky^1,4 and R. Chaloupkova¹

¹Loschmidt Laboratories, Department of Experimental Biology and Centre for Toxic Compounds in the Environment, Masaryk University, 625 00 Brno, ²Institute of Nanobiology and Structural Biology, Academy of Sciences of the Czech Republic, 373 33 Nove Hrady, ³Human Computer Interaction Laboratory, Faculty of Informatics, Masaryk University, 602 00 Brno, ⁴International Clinical Research Center, St. Anne's University Hospital, 656 91 Brnov

veronika@chemi.muni.cz, briza@chemi.muni.cz

Enzymatic transformations in organic co-solvents are being increasingly used for many applications, for example in organic synthesis of chiral compounds [1]. However, the presence of organic co-solvents in the reaction mixture generally leads to enzyme inactivation by denaturation, conformational rigidity or inhibition [2]. Detailed understanding of the effects of solvent molecules on enzyme functionality can be useful for selection of appropriate type and concentration of solvent for particular enzymatic reaction.

In the present study, the structure and function of haloalkane dehalogenases DbjA from Bradyrhizobium japonicum USDA110, DhaA from Rhodococcus rhodochrous NCIMB13064 and LinB from Sphingobium japonicum UT26, were investigated in the presence of three representative organic co-solvents: formamide 5% (v/v), acetone 20% (v/v) and isopropanol 10% (v/v). Systematic activity screening, structural analysis, molecular dynamics simulations and steady-state kinetic measurements were employed to get an insight into the mechanisms controlling the enzyme-solvent interactions at the molecular level. The results demonstrated that enzyme inactivation at high co-solvents concentrations is attributed to the conformation changes, while at lower concentrations both inhibition and activation took place. All three studied solvents were found to enter the enzymes’ access tunnels and active sites, but did not act as competitive inhibitors. At low concentrations, the co-solvents either enhanced catalysis by lowering K_0.5 or increasing k_cat, or caused enzyme inactivation by promoting substrate inhibition. A computational method, involving molecular dynamics simulations and quantitative analysis of the active sites and access tunnels occupancies by the solvent molecules, was developed. This tool can be used for prediction the effect of organic co-solvents on enzymatic activity, due to identified correlation between the portion of the active site cavity occupied by the solvent and enzyme activation or inhibition.

Our study contributes to understanding of protein-solvent interactions and demonstrates that rational selection of an optimal protein-solvent pair and effective co-solvent concentration is possible.

This work was supported by the European Regional Development Fund (CZ.1.05/2.1.00/01.0001 and CZ.1.05/1.1.00/02.0123), the Grant Agency of the Czech Republic (203/08/0114 and P207/12/0775), the Grant Agency of the Czech Academy of Sciences (IAA401630901). The access to computing facilities owned by parties and projects contributing to the MetaCentrum and listed at http://www.metacentrum.cz/acknowledgment/ is highly appreciated.

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

2. R. K. Eppler, R. S. Komor, J. Huynh, J. S. Dordick, J. A. Reimer, D. S. Clark, Proc. Nat. Aca. Sci. USA. 103, (2006), 5706-5710.