Crystal Structure of the Haloalkane Dehalogenase fromSphingomonas paucimobilisUT26^,

Abstract

The haloalkane dehalogenase from Sphingomonas paucimobilis UT26 (LinB) is the enzyme involved in the degradation of the important environmental pollutant gamma-hexachlorocyclohexane. The enzyme hydrolyzes a broad range of halogenated cyclic and aliphatic compounds. Here, we present the 1.58 A crystal structure of LinB and the 2.0 A structure of LinB with 1,3-propanediol, a product of debromination of 1,3-dibromopropane, in the active site of the enzyme. The enzyme belongs to the alpha/beta hydrolase family and contains a catalytic triad (Asp108, His272, and Glu132) in the lipase-like topological arrangement previously proposed from mutagenesis experiments. The LinB structure was compared with the structures of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 and from Rhodococcus sp. and the structural features involved in the adaptation toward xenobiotic substrates were identified. The arrangement and composition of the alpha-helices in the cap domain results in the differences in the size and shape of the active-site cavity and the entrance tunnel. This is the major determinant of the substrate specificity of this haloalkane dehalogenase.

Microbial dehalogenation

“…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

“…Secondary structure elements are indicated on the x-axis (␣-helices as the black bars, ␤-strands as the gray bars). The temperature factors for the crystal structures were extracted from PDB files (Verschueren et al, 1993b;Newman et al, 1999;Marek et al, 2000). 5E).…”

“…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).…”

“…The characteristic structural motif of haloalkane dehalogenases is a halide-binding site. The halide ion released during the dehalogenation reaction is mainly stabilized by the hydrogen bonds provided by two tryptophan residues (Verschueren et al 1993b) or by tryptophan and asparagine (Newman et al 1999;Marek et al 2000).…”