Modelling of product release and identification of export routes in haloalkane dehalogenase DhaA
Martin Klvaņa1$, Rebecca C. Wade3, Petr Kulhįnek2 and Jiųķ Damborskż1
1Loschmidt Laboratories, Faculty of Science, Masaryk University in Brno, Kamenice 5/A4, 625 00 Brno, Czech Republic
2National Centre for Biomolecular Research, Faculty of Science, Masaryk University in Brno, Kamenice 5/A4, 625 00 Brno, Czech Republic
3Molecular and Cellular Modeling Group, European Media Laboratory Research, Villa Bosch, Schloss-Wolfsbrunnenweg 33, D-69118 Heidelberg, Germany
Halogenated aliphatic hydrocarbons are widespread recalcitrant compounds due to massive natural and industrial production. They are environmentally dangerous because of their toxic, genotoxic, teratogenic and irritating effects. Microbial enzymes haloalkane dehalogenases have been studied for decades for their possible use in bioremediation owing to their capability to catalyse hydrolytic dehalogenation of the xenobiotics to harmless products. Practical use of these enzymes requires increase of activity, specificity and thermostability through modification in their structures.
Three haloalkane dehalogenases with experimentally solved three-dimensional structure differ in rate-limiting step of the dehalogenation reaction. The slowest step for Rhodoccocal haloalkane dehalogenase DhaA is release of an alcohol from the active site cavity to bulk solvent. The release may occur through two tunnels called the main tunnel and the slot. Rational computer-assisted re-design of the tunnels could lead to DhaA with higher catalytic activity. Classical molecular dynamics is, however, not appropriate tool for modelling of egress of the product of the reaction because this process is rare event and cannot be observed during nanosecond simulations. We applied Random Accelerated Molecular Dynamics (RAMD)  to speed up the egress by applying a force on the product molecule. Using RAMD, we are capable to model export pathways in the time scale from tens to hundreds picoseconds.
RAMD simulations were performed with DhaA in complex with two products of dehalogenation of 1,2,3-trichloropropane, i.e. chloride, (R)- and (S)-2,3-dichloro-1-propanol (R- and S-DCL). Complexes were prepared by molecular docking and equilibrated using classical molecular dynamics. Chloride anion solvated by water molecules left the active site through main tunnel in the presence of S-DCL after 1.7 ns of equilibration phase of molecular dynamics. Its release has never been observed in simulations without alcohol and suggested that chloride leaves the active site with the assistance of water molecules and before DCL. RAMD applied on DCL showed two pathways for DCL, i.e. the main tunnel and the slot, the tunnel being preferred export route. Based on these observations we designed directed evolution experiments attempting to improve catalytic performance of DhaA by engineering of its export routes.
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