1Department of Physical Chemistry, Faculty of Science, Palacky University Olomouc, tr. Svobody 26, 771 46 Olomouc
2National Centre for Biomolecular Research, Masaryk University, Kotlarska 2, 611 37 Brno
Biological degradation of 1,2,3-trichloropropane (TCP) is studied in the context of removal of halogenated hydrocarbon from the environment as well as from the reaction mixture formed during epichlorohydrine production. The bacterial enzyme haloalkane dehalogenase DhaA from Rhodococcus sp. (EC 18.104.22.168) transforms TCP to 2,3-dichloropropane-1-ol. This reaction step is the slowest in TCP mineralization. One possibility leading to increased activity of DhaA is modification of the enzyme by mutations. Directed evolution method resulted in two mutations (C176Y and Y273F) increasing the enzymatic activity .
The objective of this work was to explain increased enzymatic activity of DhaA mutants by molecular dynamics (MD) simulations. Molecular docking method suggested three different TCP binding modes within the DhaA active site (bm1, bm2 and bm3). Therefore, nine MD simulations of DhaA/TCP complex were run: three simulations (bm1-bm3) per each enzyme (wild type - WT-DhaA; mutant C176Y - M1-DhaA; and mutant C176Y+Y273F - M2-DhaA). The reactive center on the substrate molecule was specified as well as corresponding product for all binding modes. The bm1 is expected to lead to 1,3-dichloropropane-2-ol, bm2 to (S)-2,3-dichloropropane-1-ol and bm3 to (R)-2,3-dichloropropane-1-ol.
The TCP reorientation from bm1 to bm2 during the first 500 ps was observed in all simulation starting from bm1. Therefore, it can be concluded that bm1 is a metastable state. This observation is in a good agreement with experimental data. Furthermore, hydrogen bond interaction was observed between Tyr273 hydroxyl and Asn41 backbone carbonyl in the WT‑DhaA and M1-DhaA simulations. This interaction is not possible in M2-DhaA mutant allowing Asn41 backbone carbonyl hydrogen bond interaction with the catalytic water molecule. It causes better orientation of the catalytic water for the second reaction step (AdN addition of the catalytic water to intermediate ester group). The change in the catalytic water orientation is probably the main effect of the Y273F mutation on increased DhaA activity. The C176Y mutation causes the increase in the attraction between the residue 176 and Phe144 and decrease in the attraction between residue 176 and Lys175. The upper tunnel is one of the entrances to DhaA active site and the C176Y mutation can influence substrate, products, and/or water exchange between bulk and active site.
 T. Bosma, J. Damborsky, G. Stucki, D.B. Janssen: APPL ENVIRON MICROBIOL, 68 (2002) 3582-3587.