Balancing the Stability–Activity Trade-Off by Fine-Tuning Dehalogenase Access Tunnels

0452900 - ÚVGZ 2016 RIV DE eng J - Journal Article
Lišková, V. - Bednář, D. - Prudnikova, Tatyana - Řezáčová, P. - Koudeláková, T. - Šebestová, E. - Kutá-Smatanová, Ivana - Březovský, J. - Chaloupková, R. - Damborský, J.
Balancing the Stability–Activity Trade-Off by Fine-Tuning Dehalogenase Access Tunnels.
ChemCatChem. Roč. 7, č. 4 (2015), s. 648-659. ISSN 1867-3880. E-ISSN 1867-3899
Institutional support: RVO:67179843
Keywords : alkanes * enzymes * catalysis * halogenation * molecular dynamics * protein engineering
Subject RIV: CE - Biochemistry
Impact factor: 4.724, year: 2015

A variant of the haloalkane dehalogenase DhaA with greatly enhanced stability and tolerance of organic solvents but reduced activity was created by mutating four residues in the access tunnel. To create a stabilised enzyme with superior catalytic activity, two of the four originally modified residues were randomised. The resulting mutant F176G exhibited 32- and 10-times enhanced activity towards 1,2-dibromoethane in buffer and 40% DMSO, respectively, upon retaining high stability. Structural and molecular dynamics analyses demonstrated that the new variant exhibited superior activity because the F176G mutation increased the radius of the tunnel’s mouth and the mobility of a-helices lining the tunnel. The new variant’s tunnel was open in 48% of trajectories, compared to 58% for the wild-type, but only 0.02% for the original fourpoint variant. Delicate balance between activity and stability of enzymes can be manipulated by fine-tuning the diameter and dynamics of their access tunnels.
Permanent Link: http://hdl.handle.net/11104/0253863