Haloalkane Dehalogenases:  Structure of aRhodococcusEnzyme^,

Abstract

The hydrolytic haloalkane dehalogenases are promising bioremediation and biocatalytic agents. Two general classes of dehalogenases have been reported from Xanthobacter and Rhodococcus. While these enzymes share 30% amino acid sequence identity, they have significantly different substrate specificities and halide-binding properties. We report the 1.5 A resolution crystal structure of the Rhodococcus dehalogenase at pH 5.5, pH 7.0, and pH 5.5 in the presence of NaI. The Rhodococcus and Xanthobacter enzymes have significant structural homology in the alpha/beta hydrolase core, but differ considerably in the cap domain. Consistent with its broad specificity for primary, secondary, and cyclic haloalkanes, the Rhodococcus enzyme has a substantially larger active site cavity. Significantly, the Rhodococcus dehalogenase has a different catalytic triad topology than the Xanthobacter enzyme. In the Xanthobacter dehalogenase, the third carboxylate functionality in the triad is provided by D260, which is positioned on the loop between beta7 and the penultimate helix. The carboxylate functionality in the Rhodococcus catalytic triad is donated from E141. A model of the enzyme cocrystallized with sodium iodide shows two iodide binding sites; one that defines the normal substrate and product-binding site and a second within the active site region. In the substrate and product complexes, the halogen binds to the Xanthobacter enzyme via hydrogen bonds with the N(eta)H of both W125 and W175. The Rhodococcusenzyme does not have a tryptophan analogous to W175. Instead, bound halide is stabilized with hydrogen bonds to the N(eta)H of W118 and to N(delta)H of N52. It appears that when cocrystallized with NaI the Rhodococcus enzyme has a rare stable S-I covalent bond to S(gamma) of C187.

Steady-State and Pre-Steady-State Kinetic Analysis of Halopropane Conversion by a Rhodococcus Haloalkane Dehalogenase

“…DhaA dehalogenated mono-and dibrominated propanes with a catalytic efficiency similar to that of DhlA, whereas the catalytic efficiency of DhaA on 1,2,3-tribromopropane was ∼30-fold higher (25,26). This observation is consistent with the larger active site cavity of DhaA that can probably better accommodate large bulky substrates such as 1,2,3-tribromopropane (13). DhaA dehalogenated the different chlorinated propanes with considerable differences in maximum turnover as well as affinity.…”

“…Compared to DhlA, the active site of DhaA is connected to the solvent by a much larger tunnel and is less buried in the protein core (12). In addition, Newman et al suggested that at pH 7-9, where the enzyme activity is measured, the cap domain of DhaA becomes more flexible (13). These observations suggest that the accessibility of water to the active site may be facilitated, allowing a faster exchange of the halide ions and water with the bulk solvent.…”

Microbial dehalogenation

“…The haloalkane dehalogenases that degrade these compounds are homologous to the classical enzyme from Xanthobacter autotrophicus [3]. Even though sequence similarities are low, the overall structures are very similar and the catalytic residues are conserved [4]. Another example of a recently studied hydrolytic dehalogenase is LinB from Sphingomonas paucimobilis; this enzyme is involved in the conversion of tetrachlorocyclohexadiene, an intermediate in the γ-hexachlorocyclohexane degradation pathway ( Figure 1) [5].…”

“…The enzymes possess a catalytic triad formed by residues of the main domain, with an aspartate as the nucleophile that displaces the halogen from the substrate. In addition, these enzymes have a conserved tryptophan residue in the main domain and variable residues (phenylalanine or asparagine) in the cap domain that contribute to halide binding [4,5].…”

Functionally relevant motions of haloalkane dehalogenases occur in the specificity-modulating cap domains

“…The "static" structural determinants of the substrate specificity were implied from the crystallographic analysis of three dehalogenases belonging to different specificity classes, that is, Xanthobacter autotrophicus GJ10 (DhlA; Verschueren et al 1993b), Rhodococcus sp. (DhaA; Newman et al 1999), and Sphingomonas paucimobilis UT26 (LinB; Marek et al 2000). The substrate specificity appears to be modulated not only by the size and shape of the active site but also by the position and shape of tunnels connecting buried active sites with a bulk solvent (Damborsky and Koca 1999).…”