Pathways and Mechanisms for Product Release in the Engineered Haloalkane Dehalogenases Explored Using Classical and Random Acceleration Molecular Dynamics Simulations |
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Authors: | Martin Klvana Tana Koudelakova Pavel Dvorak Alena Stsiapanava Ivana Kuta-Smatanova Jan Dohnalek Rebecca C. Wade |
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Affiliation: | 1 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 2 Institute of Physical Biology, University of South Bohemia, Zámek 136, 373 33 Nové Hrady, Czech Republic 3 Institute of Systems Biology and Ecology, Academy of Sciences of the Czech Republic, Zámek 136, 373 33 Nové Hrady, Czech Republic 4 Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovského náměstí 2, 162 06 Praha 6, Czech Republic 5 Laboratory of Computational Chemistry, National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kamenice 5/A4, 625 00 Brno, Czech Republic 6 Molecular and Cellular Modeling Group, EML Research, Schloss-Wolfsbrunnenweg 33, D-69118 Heidelberg, Germany |
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Abstract: | Eight mutants of the DhaA haloalkane dehalogenase carrying mutations at the residues lining two tunnels, previously observed by protein X-ray crystallography, were constructed and biochemically characterized. The mutants showed distinct catalytic efficiencies with the halogenated substrate 1,2,3-trichloropropane. Release pathways for the two dehalogenation products, 2,3-dichloropropane-1-ol and the chloride ion, and exchange pathways for water molecules, were studied using classical and random acceleration molecular dynamics simulations. Five different pathways, denoted p1, p2a, p2b, p2c, and p3, were identified. The individual pathways showed differing selectivity for the products: the chloride ion releases solely through p1, whereas the alcohol releases through all five pathways. Water molecules play a crucial role for release of both products by breakage of their hydrogen-bonding interactions with the active-site residues and shielding the charged chloride ion during its passage through a hydrophobic tunnel. Exchange of the chloride ions, the alcohol product, and the waters between the buried active site and the bulk solvent can be realized by three different mechanisms: (i) passage through a permanent tunnel, (ii) passage through a transient tunnel, and (iii) migration through a protein matrix. We demonstrate that the accessibility of the pathways and the mechanisms of ligand exchange were modified by mutations. Insertion of bulky aromatic residues in the tunnel corresponding to pathway p1 leads to reduced accessibility to the ligands and a change in mechanism of opening from permanent to transient. We propose that engineering the accessibility of tunnels and the mechanisms of ligand exchange is a powerful strategy for modification of the functional properties of enzymes with buried active sites. |
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Keywords: | CC loop, C-terminal cap domain loop (α7/α8 loop) CL, chloride anion DCL, 2,3-dichloropropane-1-ol TCP, 1,2,3-trichloropropane MD, molecular dynamics NC loop, N-terminal cap domain loop (β6/α4 loop) RAMD, random acceleration molecular dynamics PDB, Protein Data Bank |
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