Crystal structure of the probable haloacid dehalogenase PH0459 from Pyrococcus horikoshii OT3

Abstract

PH0459, from the hyperthermophilic archaeon Pyrococcus horikoshii OT3, is a probable haloacid dehalogenase with a molecular mass of 26,725 Da. Here, we report the 2.0 A crystal structure of PH0459 (PDB ID: 1X42) determined by the multiwavelength anomalous dispersion method. The core domain has an alpha/beta structure formed by a six-stranded parallel beta-sheet flanked by six alpha-helices and three 3(10)-helices. One disulfide bond, Cys186-Cys212, forms a bridge between an alpha-helix and a 3(10)-helix, although PH0459 seems to be an intracellular protein. The subdomain inserted into the core domain has a four-helix bundle structure. The crystal packing suggests that PH0459 exists as a monomer. A structural homology search revealed that PH0459 resembles the l-2-haloacid dehalogenases l-DEX YL from Pseudomonas sp. YL and DhlB from Xanthobacter autotrophicus GJ10. A comparison of the active sites suggested that PH0459 probably has haloacid dehalogenase activity, but its substrate specificity may be different. In addition, the disulfide bond in PH0459 probably facilitates the structural stabilization of the neighboring region in the monomeric form, although the corresponding regions in l-DEX YL and DhlB may be stabilized by dimerization. Since heat-stable dehalogenases can be used for the detoxification of halogenated aliphatic compounds, PH0459 will be a useful target for biotechnological research.

Figure 1.

(A) Sequence alignment of homologs of the PH0459 protein. The alignment was generated by CLUSTALW (Thompson et al. 1994) and ESPript (Gouet et al. 1999). The secondary structures of PH0459, as determined by DSSP (Kabsch and Sander 1983), are shown above the sequences (α, α-helix; β, β-strand; η, 3₁₀-helix; T, β-turn). (B) Ribbon representations of the PH0459 structure (stereo view). (Color gradient [blue, green, yellow, red] from N terminus to C terminus.) The disulfide bond, Cys186–Cys212, is shown in a stick model.

Figure 2.

(A) The superimposition of the main-chain structures of PH0459 (green), l-DEX YL (magenta) (PDB ID: 1JUD), and DhlB (orange) (PDB ID: 1QQ5) monomers. The disulfide bond in PH0459 is shown in a sphere model. (B) The superimposition of the main-chain structures of the PH0459 (green) monomer, the l-DEX YL (magenta), and DhlB (orange) dimers. The disulfide bond in PH0459 is shown in a sphere model. (C) The superimposition of the active sites of PH0459 (green), l-DEX YL (magenta), and DhlB (orange) (stereo view). (D) Surface representations of PH0459, l-DEX YL, and DhlB. The active site residues are colored yellow. The structures are oriented in the same direction.