Haloalkane dehalogenases (EC 18.104.22.168) are key enzymes for the microbial degradation of halogenated aliphatic compounds that occur as soil pollutants (Janssen et al.
; Oberg, 2002
; Ballschmiter, 2003
; Janssen, 2004
). The enzymes catalyze the hydrolysis of a carbon–halogen bond in halogenated substrates, producing a corresponding alcohol, a halide ion and a proton. To date, the crystal structures of three haloalkane dehalogenases have been determined: those of DhlA from Xanthobacter autorophicus
GJ10 (Verschueren et al.
), DhaA from Rhodococcus rhodochrous
NCIMB 13064 (Newman et al.
) and LinB from Sphingobium japonicum
UT26 (Marek et al.
). These crystal structures revealed that the three enzymes essentially adopt the same fold and are composed of two domains: the main and cap domains. The active site of the enzyme is in an occluded cavity located between the two domains.
In order to design novel haloalkane dehalogenases that can catalyze the hydrolysis of recalcitrant environmental pollutants, intensive protein-engineering studies have been carried out on haloalkane dehalogenases. Random mutagenesis of DhaA (Bosma et al.
) and site-directed mutagenesis of LinB (Chaloupkova et al.
) are the only examples that have successfully changed the substrate specificity of the haloalkane dehalogenase. However, since changing the substrate specificity using protein-engineering techniques remains difficult, the isolation and characterization of new haloalkane dehalogenases are still necessary in order to develop enzymes with unique substrate specificity.
In the 1990s, haloalkane dehalogenases were thought to only be present in soil bacteria that colonize contaminated environments (Fetzner & Lingens, 1994
). Recent progress in the genome-sequence analyses of various bacteria, however, has revealed that more than 20 bacteria that colonize unpolluted environments have open reading frames (ORFs) encoding putative dehalogenases (Jesenska et al.
). These ORF products should be useful as a genetic source to isolate new haloalkane dehalogenases. We focused on the genome sequence of Bradyrhizobium japonicum
USDA110 (Kaneko et al.
) and succeeded in isolating a haloalkane dehalogenase with unique substrate specificity. The haloalkane dehalogenase was named DbjA (Sato et al.
). DbjA has 25% identity to DhlA, 49% identity to DhaA and 41% identity to LinB. DbjA has typical haloalkane dehalogenase activity and a high catalytic activity for β-methylated haloalkanes (Sato et al.
). It is of note that β-methylated haloalkanes are scarcely hydrolyzed by most haloalkane dehalogenases, probably owing to the steric hindrance of the branched methyl group. A sequence comparison between DbjA and other haloalkane dehalogenases has suggested that an 11-amino-acid insertion between the main and cap domains of DbjA produces a unique active-site structure that results in the unique substrate specificity of DbjA (Sato et al.
). To confirm this hypothesis on the basis of the three-dimensional structure of DbjA, we initiated crystal structure analysis of the enzyme. Here, we report the crystallization of DbjA.