Engineering the activity of haloalkane dehalogenase with toxic synthetic substrate using methods of focused directed evolution

P. Dvořák, M. Pavlová, M. Klvaňa, J. Brezovský, R. Chaloupková, Z. Prokop,

and J. Damborský

Loschmidt laboratories, Institute of Experimental Biology and National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kamenice 5/A4, 625 00 Brno, Czech Republic (paveld@chemi.muni.cz)

Haloalkane dehalogenases (HLDs; EC 3.8.1.5.) are microbial enzymes that catalyze hydrolytic conversion of haloalkanes to the corresponding alcohols and hydrogen halides [1]. Some synthetic halogenated alkanes, like 1,2-dichloroethane (DCE), are produced worldwide and are classified as persistent environmental pollutants. DCE is widely used as solvent and intermediate in chemical industry. Its toxic and carcinogenic effects are well recognized. HLDs have been investigated for possible applications in detoxification of halogenated pollutants including DCE [2,3]. It remains a challenge to provide HLDs that could degrade these substrates efficiently. Here we report construction of a mutant dehalogenase with improved catalytic activity towards DCE.

Dehalogenase DhaA from Rhodococcus erythropolis Y2 [4] has only minimal activity with DCE. Large cavity of DhaA active site and wide mouth of the main tunnel cannot effectively accommodate small DCE molecule and results in non-productive binding. We hypothesized that reduction of size of DhaA active site and tunnels together with elimination of non-productive binding could lead to the increased activity with DCE.

Initially, computer modeling, site-directed and saturation mutagenesis were applied to target the passage of ligands through the access tunnels [5]. Up to seven bulky substitutions (I135F, A145F, A172F, C176Y, V245F, L246I, Y273F) were introduced into the walls of DhaA tunnels and in vicinity of the active site. Closing up the tunnels influenced the substrate specificity of DhaA and improved the enzyme’s activity towards DCE 8-times. This DhaA variant was used as a template for the second round of in vitro evolution. During this second step four residues (I132, L209, F245 and I246) were randomized with the aim to reduce the size of DhaA active site and to influence the non-productive binding of DCE above the catalytic histidine. Screening of four thousand clones from resulting mutant libraries revealed one positive DhaA variant (F245T+I246L) which showed 12-times higher specific activity with DCE compared to the wild-type DhaA.

The substitutions showing the most significant impact on DhaA activity with DCE will be inserted to another HLD to validate their effect. Understanding the structure-function relationships in constructed mutants is important to design next generation of biocatalysts for degradation of anthropogenic substrate.

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