Engineering enzyme access tunnels for protein stabilization

Veronika Liskova, David Bednar, Tana Koudelakova, Eva Sebestova, Jan Brezovsky,
Jiri Damborsky, and Radka Chaloupkova

Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, International Centre for Clinical Research, St. Anne's University Hospital in Brno, Pekarska 53, 656 91, Brno

verca.liskova@centrum.cz

Enzymes have a great potential in many areas of biotechnology. Natural enzymes have not evolved for hostile industrial environment such as extreme pH, elevated temperature or presence of co-solvents, thus stabilization of enzymes against unnatural conditions has become an important target for protein engineers. Most of the studies focused on enzyme stabilisation focused on active site or its vicinity, on modification of protein surface or highly flexible protein regions [1-6]. Haloalkane dehalogenases are predominantly microbial enzymes with great potential in biotechnology and biocatalysis. However, these practical applications are limited due to low resistance of haloalkane dehalogenases to organic co-solvents and elevated temperature [7]. Recently constructed variant of haloalkane dehalogenase DhaA exhibited 4000-fold improved kinetic stability in 40 % (v/v) DMSO, enhanced thermostability by 16.4 °C, but 100-fold lower catalytic activity with 1,2-dibromoethane in a pure buffer compared to the wild type enzyme. Enzyme stabilisation was achieved by introduction of four bulkier and mostly hydrophobic residues into the enzyme access tunnel. These residues improved a contact with other residues of the access tunnel, enhanced packing of hydrophobic core and prevented the entry of DMSO into the active-site cavity [8]. Objective of this follow up study was focused on improvement of catalytic activity of highly stable DhaA variant in pure buffer with minimum loss of its stability. Saturation mutagenesis in two of the four tunnel positions resulted in a single point variant, whose catalytic activity was increased 32- and 10-fold in pure buffer and in 40 (v/v) % DMSO, respectively, while thermal stability was lowered just by 4 °C. Structural analysis and molecular dynamics revealed that the newly introduced mutation (F176G) reopened previously closed tunnel in stable DhaA and thus restored enzymatic activity, while remaining tunnel mutations maintained protein stability.Optimization of amino acid residues lining the access tunnels thus represents a generally-applicable strategy for fine-tuning stability-function trade-off of enzymes with buried active sites.

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