Engineering tunnels in enzymes

A. Gora¹, J. Brezovsky¹, K. Hasan¹, A. Fortova¹, J. Damborsky^1,2

¹Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic

²International Centre for Clinical Research, St. Anne's University Hospital Brno, Pekarska 53, 656 91 Brno, Czech Republic

artgora@gmail.com

Enzymes are natural biocatalysts evolved for high selectivity and activity towards a wide range of substrates. Protein engineering makes a use of the knowledge gained from studies of protein structure/reactivity/selectivity relationships to construct improved biocatalysts for practical applications. Modifications of the residues forming the first or second shell of an active site are currently the most commonly used for successful design of constructs with a higher activity and selectivity, improved protein stability, or broadened substrate specificity. However, many enzymes show significant changes in their properties also due to mutations in the positions far from the active site [1]. Herein we present two examples of enzyme engineering focused on the residues forming the tunnels and their gates, as a new strategy for efficient control of enzyme properties.

DatA and LinB are examples of the haloalkane dehalogenases with buried active site. Their activity and selectivity reflects the properties of the substrate/product transport pathways. In this project, we have proposed mutations for the modification of the access pathways of these two enzymes using a novel approach. The rational design of mutants was assisted by the CAVER 3.0 software [2], which is the computational tools for the analysis of tunnels in static structures as well as in their ensembles from molecular dynamics simulations. The opening of the access tunnel in the DatA enzyme was shown to increase enzyme activity, whereas the closing of the main access pathway in LinB enzyme decreased it significantly. Moreover, the designed mutants showed also highly altered substrate specificity. Our work provides evidence that the careful redesign of access pathways presents a powerful strategy for the precise control of the activity and selectivity of enzymes.

This work was financially supported by the European Regional Development Fund (CZ.1.05/2.1.00/01.0001 and CZ.1.05/1.1.00/02.0123), by the Czech Grant Agency (203/08/0114 and P503/12/0572), and the Grant Agency of the Czech Academy of Sciences (IAA401630901). The work of A.G. was supported by SoMoPro programme No. SIGA762 and has received a financial contribution from the E.C. within the 7th FP (FP/2007-2013) under grant agreement No. 229603 and is co-financed by the South Moravian Region.

[1] J. Lee& N.M. Goodey, Chem. Rev., 111, (2011), 7595.

[2] E. Chovancova, A. Pavelka, P. Benes, O. Strnad, J. Brezovsky, B. Kozlikova, A. Gora, V. Sustr, M. Klvana, P. Medek, L. Biedermannova, J. Sochor, J. Damborsky, CAVER 3.0, in preparation.