Engineering Enzyme Resistance to Organic Co-Solvent

T. Koudeláková¹, R. Chaloupková¹, J. Brezovský¹, Z. Prokop¹, M. Pavlová¹, M. Hesseler², M. Khabiri³, R. Ettrich³, U. T. Bornscheuer² and J. Damborský¹

¹Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment, Masaryk University, Kamenice 5/A13, 62500 Brno, Czech Republic

²Department of Biotechnology and Enzyme Catalysis, Ernst Moritz Arndt University, Felix-Hausdorff-Str. 4, D-17487 Greifswald, Germany

³Institute of Nanobiology and Structural Biology, Academy of Sciences of the Czech Republic, Zámek 136, 373 33 Nové Hrady, Czech Republic

tangerine@chemi.muni.cz

Haloalkane dehalogenases (EC 3.8.1.5) are representatives of the α/β-hydrolase fold superfamily [1]. These enzymes can hydrolytically cleave a halogen atom from more than hundred chlorinated, brominated or iodinated alkanes and their derivatives [2-3]. Access of halogenated substrates to the buried active site, and egress of formed alcohols and halides are facilitated by the protein tunnels [4]. The industrial use of these enzymes is limited by their instability in the presence of co-solvents, which are used for solubilization of hydrophobic substrates. Here we present a systematic protein engineering study, which examines features determining resistance of haloalkane dehalogenases towards organic co-solvent dimethyl sulfoxide. The initial random mutagenesis of a dehalogenase gene followed by a colorimetric screening in 42% dimethyl sulfoxide revealed importance of the residues lining the access tunnels for protein stability. Substitutions introduced to the residues lining the main access tunnel led to the enzyme variants with improved kinetic stability, increased structural resistance towards dimethyl sulfoxide and elevated structural thermostability. The experimental results were supported by molecular dynamics simulations. Based on our findings, the resistance of haloalkane dehalogenases and possibly also other enzymes with buried active sites can be improved by modification of their access tunnels.

The work was supported by grants LC06010, IAA401630901, MSM0021622412 and CZ.1.05/2.1.00/01.0001. FEMS and Ernst Moritz Arndt University are acknowledged for financial support of research fellowships of TK.

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