Abstract
Distinct mechanisms have been proposed for the biological dehalogenation catalyzed by cobalamin-dependent enzymes, with two recent crystallographic studies suggesting different mechanisms based on the observed interaction between the organohalide substrate and cobalamin. In one case, involving an aromatic dibromide substrate in NpRdhA, a novel CoII-Br interaction was observed using EPR, suggesting a mechanism involving a [CoXR] adduct. However, in the case of trichloroethylene in PceA, a significantly longer Co-Cl distance was observed in X-ray crystal structures, suggesting a dissociative electron transfer mechanism. Subsequent DFT models of these reactions have not reproduced these differences in binding modes. Here, we have performed molecular docking and DFT calculations to investigate and compare the interaction between different organohalides and cobalamin in both NpRdhA and PceA. In each case, despite differences in binding in the CoII state, the reaction likely proceeds via formation of a [CoXR] adduct in the CoI state that weakens the breaking carbon-halide bond, suggesting this could be a general mechanism for cobalamin-dependent dehalogenation.
Highlights
Organohalide-respiring bacteria are responsible for the biological degradation of a range of organohalide pollutants such as polychlorinated biphenyls and dioxins.[1,2,3,4] Reductive dehalogenases (RDase) overcome the inherent difficulty of direct nucleophilic substitution of halide from aryl or vinyl substrates by concomitantly reducing the substrate using a base-off cobalamin cofactor
Transition states were obtained by relaxed potential energy scans along the distance between the C bound to the transferring X and the H of the active site Tyr, with a step size of no more than 0.03 Å near the maximum
Docking dibromo-4-hydroxybenzoic acid (DBHB) into NpRdhA required the inclusion of four flexible residues, so it appears easier to achieve binding with TCE in PceA than with DBHB in NpRdhA
Summary
Organohalide-respiring bacteria are responsible for the biological degradation of a range of organohalide pollutants such as polychlorinated biphenyls and dioxins.[1,2,3,4] Reductive dehalogenases (RDase) overcome the inherent difficulty of direct nucleophilic substitution of halide from aryl or vinyl substrates by concomitantly reducing the substrate using a base-off cobalamin cofactor. Two [4Fe–4S] clusters are responsible for reducing the cob(II)alamin resting state to the active cob(I)alamin.[5] Two recent X-ray crystallographic studies of the NpRdhA and PceA enzymes reveal the RDase active site contains a conserved Tyr ideally positioned above the cobalamin for protonation of the substrate during the reaction, facilitated by an adjacent positively charged Arg or Lys residue that stabilises the deprotonated Tyr.[6,7] differences in the position of the substrate/product bound within the active site of the resting states of NpRdhA and PceA have led to distinct mechanistic proposals for each enzyme, and subsequent DFT calculations used to analyse these mechanisms has not reproduced these distinct binding modes.[8,9].
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