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Redesigning dehalogenase access tunnels as a strategy for degrading an anthropogenic substrate

Nature Chemical Biology volume 5, pages 727–733 (2009)Cite this article

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.

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Figure 1: Hot spot residues lining the access tunnels selected for mutagenesis by computer simulations.
Figure 2: Characteristics of mutants with enhanced activity against TCP.
Figure 3: Structural basis of enhanced activity against TCP.

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Acknowledgements

We thank V. de Lorenzo (Centro Nacional de Biotecnologia, Spain) and U. Bornscheuer (University Greifswald, Germany) for critical reading of this manuscript. R.C.W. gratefully acknowledges the support of the Klaus Tschira Foundation and M.P. acknowledges a scholarship from the Japan Society for Promotion of Science. We acknowledge financial support from the Ministry of Education of the Czech Republic (LC06010 and MSM0021622412), the Grant Agency of the Czech Republic (201/07/0927 and 203/08/0114), the Grant Agency of the Czech Academy of Sciences (IAA401630901), the North Atlantic Treaty Organization (EST.CLG.980504), and Grants-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology, Japan and the Ministry of Agriculture, Forestry, and Fisheries, Japan. We acknowledge the Supercomputing Centre Brno for computational resources.

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Author notes

  1. Martina Pavlova and Martin Klvana: These authors contributed equally to this work.

Authors and Affiliations

  1. Loschmidt Laboratories, Institute of Experimental Biology and National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Brno, Czech Republic

    Martina Pavlova, Martin Klvana, Zbynek Prokop, Radka Chaloupkova & Jiri Damborsky

  2. Department of Physical Chemistry and Center for Biomolecules and Complex Molecular Systems, Palacký University, Olomouc, Czech Republic

    Pavel Banas & Michal Otyepka

  3. Molecular and Cellular Modeling Group, EML Research gGmbH, Heidelberg, Germany

    Rebecca C Wade

  4. Department of Environmental Life Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Japan

    Masataka Tsuda & Yuji Nagata

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Contributions

M.P. performed mutagenesis and activity measurements; M.K. performed molecular modeling and designed mutants; Z.P. performed pre-steady state kinetics measurements; R.C. performed CD spectroscopy measurements and solvent kinetic isotopic effect measurements; P.B. and M.O. designed mutants; R.C.W. contributed the RAMD modeling tool; M.T. and Y.N. contributed molecular biology tools; J.D. interpreted data and designed mutants. All authors contributed to the writing of the paper.

Corresponding author

Correspondence to Jiri Damborsky.

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Pavlova, M., Klvana, M., Prokop, Z. et al. Redesigning dehalogenase access tunnels as a strategy for degrading an anthropogenic substrate. Nat Chem Biol 5, 727–733 (2009). https://doi.org/10.1038/nchembio.205

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