Mechanism-Based Biodegradability Prediction of Dehalogenase

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The deduced amino acid sequence of LinB (1, 3, 4, 6-tetrachloro-1, 4-cyclohexadiene halidohydrolase) showed highest level, of similarity to haloalkane dehalogenase (DhlA) from Xanthobacter autotrophicus GJIO, suggesting that LinB belongs to the family of haloalkane dehalogenase enzymes catalyzing the dehalogenation by hydrolytic mechanism. LinB is believed to belong the family of α/β-hydrolases which employ catalytic triad, i.e. nucleophile-histidine-acid, during the catalytic reaction. At first, the position of the catalytic triad within the sequence of LinB was probed by site-directed mutagenesis. The catalytic triad residues of the haloalkane dehalogenase LinB are proposed to be ?D108, H272, and E132 For further analysis, we determined three dimentional structure of LinB. The purified LinB was crystallized using the hanging-drop vapoul-diffusion method. The crystals diffract to 1.58 angstrom using synchrotron X-ray under cryogenic (100K) conditions. The structure of LinB was solved by molecular replacement with dehalogenase (DhaA) from Rhodococcus.