Haloalkane dehalogenases (HLDs, EC 3.8.1.5) are bacterial enzymes with α/β-hydrolase fold, which catalyse hydrolytic conversion of a broad range of halogenated aliphatic hydrocarbons into three reaction products: an alcohol, a halide anion and a proton. HLDs catalyse the reactions of great environmental and biotechnological significance with potential application in bioremediation, biosensing, decontamination of warfare agents, synthesis of optically pure compounds, cellular imaging and protein tagging [1]. However, their use in these applications is limited by their low stability and activity under the harsh conditions. 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 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. Introduced residues improved a contact with other residues of the access tunnel, enhanced packing of hydrophobic core and prevented entry of DMSO into the active-site cavity [2].
Herein presented study aimed to improve catalytic activity of the highly stable DhaA in buffer, with minimum loss of its stability. Systematic mutagenesis of two of the four originally modified tunnel residues (F176 and V172) resulted in a single point variant F176G possessing 32- and 10-times improved catalytic activity in buffer and in 40 % (v/v) DMSO, respectively. Thermostability of the mutant was lowered by 4 °C only. Moreover, the newly evolved variant exhibited enhanced activity towards 26 out of 30 tested halogenated compounds similarly to wild-type enzyme. Structural analysis and molecular dynamics revealed that newly introduced mutation F176G reopened previously closed tunnel in stable DhaA and increase the mobility of the two α-helices lining the tunnel, thus restoring the enzyme activity, while remaining tunnel mutations maintained its stability. Fine-tuning of amino acid residues lining the access tunnels thus represents generally-applicable strategy for minimisation of stability-function trade-off of enzymes with buried active sites [3].