Investigation of gating mechanisms in the access tunnel of haloakane dehalogenases

S. M. Marques, J. Damborsky and J. Brezovsky

Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment, Faculty of Science, Masaryk University, Kamenice 5/A13, 625 00 Brno, Czech Republic
smarques@mail.muni.cz

Molecular gates are structural features present in many diverse biological systems, namely enzymes, ion channels, protein-protein and protein-nucleic acid complexes. In enzymes, gates may control the ligands’ entry to and egress from buried active sites, regulate the access of solvent molecules, or even synchronize molecular events taking place in different parts of the protein. However, the mechanism of gating is not well understood in spite of their broad occurrence [1], and their rational engineering is challenging [2].

The haloalkane dehalogenases (HLDs) are bacterial enzymes that catalyze the hydrolysis of a wide variety of halogenated organic compounds into the corresponding alcohols. This property makes them very interesting for a number of biotechnological applications, such as bioremediation, biocatalysis, and biosensing. Redesign of dehalogenase tunnels has been accomplished in previous works and has proven successful to increase enzyme activity, enantioselectivity and stability. DhaA31 mutant is an example in which the narrowing of the access tunnels increased k_cat by 32-fold towards an anthropogenic pollutant, 1,2,3-trichloropropane (TCP) [3].

In the present work we investigated eventual gating processes present in the access tunnels of the DhaA31 mutant [4]. We have performed four 1 μs molecular dynamics simulations using AMBER 12 [5] and analyzed tunnel dynamics by CAVER 3.0 [6]. From these calculations, a gating process could be identified on the main tunnel of DhaA31. This gating is ruled by an alpha helix movement, associated with the movement of the side chains of key residues Phe149, Phe168, Ala172, and Tyr176. The observed gate may regulate not only the exchange of the ligands, but also the number of solvent molecules accessing the active site during the catalytic cycle. Periodic desolvation may explain the observed increase in the catalytic activity for DhaA31 with respect to the wild type. Furthermore, these studies point out the direction for additional improvements in the next round of protein engineering by a rational re-design of this gate. 

Authors gratefully acknowledge the National Sustainability Programme (LO1214) and the Czech Ministry of Education (CZ.1.07/2.3.00/30.0037) for financial support.

[1] A. Gora, J. Brezovsky, J. Damborsky, Chem. Rev. 2013, 113, 5871-5923.

[2] J. Damborsky, J. Brezovsky, Curr. Opin. Chem. Biol. 2014, 19, 8-16.

[3] M. Pavlova, M. Klvana, Z. Prokop, R. Chaloupkova, P. Banas, M. Otyepka, R. C. Wade, M. Tsuda, Y. Nagata, J. Damborsky, Nat. Chem. Biol. 2009, 5, 727-733.

[4] M., Lahoda, J. R., Mesters, A., Stsiapanava, R., Chaloupkova, M., Kuty, J., Damborsky, I., Kuta Smatanova, Acta Cryst. 2014, D70, 209-217.

[5] D. A. Case, T. A. Darden, T. E. Cheatham, III, C. L. Simmerling, J. Wang, R. E. Duke, R. Luo, R. C. Walker, W. Zhang, K. M. Merz, et al., AMBER 12, University Of California, San Francisco, 2012

[6] E. Chovancova, A. Pavelka, P. Benes, O. Strnad, J. Brezovsky, B. Kozlikova, A. Gora, V. Sustr, M. Klvana, P. Medek, et al., PLoS Comput. Biol. 2012, 8, e1002708.