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Redesigning dehalogenase access tunnels as a strategy for degrading an anthropogenic substrate
Martina Pavlova1,5, Martin Klvana1,5, Zbynek Prokop1, Radka Chaloupkova1, Pavel Banas2, Michal Otyepka2, Rebecca C Wade3, Masataka Tsuda4, Yuji Nagata4 & Jiri Damborsky1
Abstract
Engineering enzymes to degrade anthropogenic compounds efficiently is challenging. We obtained Rhodococcus rhodochrous haloalkane dehalogenase mutants with up to 32-fold higher activity than wild type toward the toxic, recalcitrant anthropogenic compound 1,2,3-trichloropropane (TCP) using a new strategy. We identified key residues in access tunnels connecting the buried active site with bulk solvent by rational design and randomized them by directed evolution. The most active mutant has large aromatic residues at two out of three randomized positions and two positions modified by site-directed mutagenesis. These changes apparently enhance activity with TCP by decreasing accessibility of the active site for water molecules, thereby promoting activated complex formation. Kinetic analyses confirmed that the mutations improved carbon-halogen bond cleavage and shifted the rate-limiting step to the release of products. Engineering access tunnels by combining computer-assisted protein design with directed evolution may be a valuable strategy for refining catalytic properties of enzymes with buried active sites.
1 Loschmidt Laboratories, Institute of Experimental Biology and National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Brno, Czech Republic.
2 Department of Physical Chemistry and Center for Biomolecules and Complex Molecular Systems, Palacky University, Olomouc, Czech Republic.
3 Molecular and Cellular Modeling Group, EML Research gGmbH, Heidelberg, Germany.
4 Department of Environmental Life Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Japan.
5 These authors contributed equally to this work.
