Conformational changes allow processing of bulky substrates by a haloalkane dehalogenase with a small and buried active site

Abstract

Haloalkane dehalogenases catalyze the hydrolysis of halogen-carbon bonds in organic halogenated compounds and as such are of great utility as biocatalysts. The crystal structures of the haloalkane dehalogenase DhlA from the bacterium from Xanthobacter autotrophicus GJ10, specifically adapted for the conversion of the small 1,2-dichloroethane (DCE) molecule, display the smallest catalytic site (110 ų) within this enzyme family. However, during a substrate-specificity screening, we noted that DhlA can catalyze the conversion of far bulkier substrates, such as the 4-(bromomethyl)-6,7-dimethoxy-coumarin (220 ų). This large substrate cannot bind to DhlA without conformational alterations. These conformational changes have been previously inferred from kinetic analysis, but their structural basis has not been understood. Using molecular dynamic simulations, we demonstrate here the intrinsic flexibility of part of the cap domain that allows DhlA to accommodate bulky substrates. The simulations displayed two routes for transport of substrates to the active site, one of which requires the conformational change and is likely the route for bulky substrates. These results provide insights into the structure-dynamics function relationships in enzymes with deeply buried active sites. Moreover, understanding the structural basis for the molecular adaptation of DhlA to 1,2-dichloroethane introduced into the biosphere during the industrial revolution provides a valuable lesson in enzyme design by nature.

Keywords: active site; conformational change; dichloroethane degradation; enzyme catalysis; enzyme kinetics; enzyme mechanism; ethylene dichloride; haloalkane dehalogenase; molecular dynamics; molecular evolution; organic halogen; organohalogen; protein conformation.

Figure 1.

The scheme of halide ion binding. The upper route starts with a slow enzyme isomerization step, after which the ion binds rapidly. The lower pathway involves a rapid binding into an initial collision complex followed by an induced slow isomerization step. The scheme was adapted from the papers by Schanstra and Janssen (11) and Krooshof et al. (12), where E represents enzyme in closed conformation; E° enzyme in open conformation; X⁻ halide ion (Br⁻ or Cl⁻); E.X⁻, E°.X⁻, and (E.X⁻)* represent enzyme–halide bound complexes. This figure was adapted from work that was originally published in Biochemistry and Protein Science. Schanstra, J. P., and Janssen, D. B. Kinetics of halide release of haloalkane dehalogenase: Evidence for a slow conformational change. Biochemistry. 1996; 35:5624–5632. ©the American Chemical Society and Krooshof, G. H., Floris, R., Tepper, A. W., and Janssen, D. B. Thermodynamic analysis of halide binding to haloalkane dehalogenase suggests the occurrence of large conformational changes. Protein Sci. 1999; 8:355–360 ©the Protein Society.

Figure 2.

The volume of the catalytic site of DhlA (PDB ID 2YXP) compared with the van der Waals volume of the two substrates. Residues 156–196 of the cap domain are colored cyan in the DhlA structure. The volume of the DhlA catalytic site was calculated using Castp web server (43) and the van der Waals volume of the ligands using the methodology by Zhao et al. (60).

Figure 3.

The two substrate binding routes explored by COU molecules in the accelerated MD simulations. On the left, a single molecule is bound to the entrance of the main tunnel p1 of HLDs and on the right two molecules are bound to p3a and p3b locations in the tunnel p3 which has been opened by a conformational change by the cap domain residues shown in cyan. The other three to four molecules are interacting with different parts of the protein surface or stacking together. The halide stabilizing Trp-125 and Trp-175 and the catalytic Asp-124 are shown as sticks to illustrate the active site.

Figure 4.

The different orientations of the protonated Glu-56 adopted during the simulations. In the blue orientation it forms the hydrogen bond with Val-219 backbone oxygen (orange dotted line) and in the pink orientation, it faces the active site depicted by the residues Trp-125, Trp-175, and Asp-124 (white sticks).

Figure 5.

Structural alignment of the open and closed conformations of DhlA. The open (lime) and closed (white) conformations of DhlA and the locations explored by the substrate DCE (black sticks) during the adaptive sampling simulation of DhlA and the deprotonated Glu-56. The substrate can leave/rebind through p1 and p3 (p3a and p3b).