In Pseudomonas paucimobilis UT26, y-hexachlorocyclohexane (-y-HCH) is converted by two steps of dehydrochlorination to a chemically unstable intermediate, 1,3,4,, which is then metabolized to 2,5-dichloro-2,5-cyclohexadiene-1,4-diol (2,5-DDOL) by two steps of hydrolytic dehalogenation via the chemically unstable intermediate 2,4,5-trichloro-2,5-cyclohexadiene-1-ol (2,4,5-DNOL). To clone a gene encoding the enzyme responsible for the conversion of the chemically unstable intermediates 1,4-TCDN and 2,4,5-DNOL, a genomic library of P. paucimobilis UT26 was constructed in Pseudomonas putida PpYlOlLA into which the linA gene had been introduced by TnS. An 8-kb BglII fragment from one of the cosmid clones, which could convert y-HCH to 2,5-DDOL, was subcloned, and subsequent deletion analyses revealed that a ca. 1.1-kb region was responsible for the activity. Nucleotide sequence analysis revealed an open reading frame (designated the linB gene) of 885 bp within the region. The deduced amino acid sequence of LinB showed significant similarity to hydrolytic dehalogenase, DhlA (D. B. Janssen, F. Pries, J. van der Ploeg, B. Kazemier, P. Terpstra, and B. Witholt, J. Bacteriol. 171:6791-6799, 1989). The protein product of the linB gene was 32 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Not only 1-chlorobutane but also 1-chlorodecane (CIO) and 2-chlorobutane, which are poor substrates for other dehalogenases, were good substrates for LinB, suggesting that LinB may be a member of haloalkane dehalogenases with broad-range specificity for substrates.

Chitosanase from Bacillus circulans MH-K1 is a 29-kDa extracellular protein composed of 259 amino acids. The crystal structure of chitosanase from B. circulans MH-K1 has been determined by multiwavelength anomalous diffraction method and refined to crystallographic R ‫؍‬ 19.2% (R free ‫؍‬ 23.5%) for the diffraction data at 1.6-Å resolution collected by synchrotron radiation. The enzyme has two globular upper and lower domains, which generate the active site cleft for the substrate binding. The overall molecular folding is similar to chitosanase from Streptomyces sp. N174, although there is only 20% identity at the amino acid sequence level between both chitosanases. However, there are three regions in which the topology is remarkably different. In addition, the disulfide bridge between Cys 50 and Cys 124 joins the ␤1 strand and the ␣7 helix, which is not conserved among other chitosanases. The orientation of two backbone helices, which connect the two domains, is also different and is responsible for the differences in size and shape of the active site cleft in these two chitosanases. This structural difference in the active site cleft is the reason why the enzymes specifically recognize different substrates and catalyze different types of chitosan degradation.

Aleuria aurantia possesses a fucose-specific lectin (AAL) that is widely used as a specific probe for fucose. Fucosylated sugars often play pivotal roles in many cellular processes. We have determined the crystal structure of AAL at 2.24 A resolution in complex with only three fucose molecules in its five sugar binding sites of a six-fold beta-propeller structure. Very recently, the structure of AAL has been independently determined, showing that all the five binding sites were occupied by fucose molecules [Wimmerova, M., et al. (2003) J. Biol. Chem. 278, 27059-27067]. Stabilization of the arginine conformation bound to fucose molecules plays an essential role in generating the difference in the affinity in the five binding sites. Binding models with a couple of saccharides based on biochemical assays suggest that hydrophobic contacts also play important roles in AAL recognizing its ligand.