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Xylan-1,4-ß-xylosidase (ß-xylosidase) hydrolyses xylooligomers at their non-reducing ends into individual xylose units. Recently, XylC, a ß-xylosidase from Thermoanaerobacterium saccharolyticum JW/SL-YS485, was found to be structurally different from corresponding glycosyl hydrolases in the CAZy database (http://www.cazy.org/), and was subsequently classified as the first member of a novel family of glycoside hydrolases (GH120). In the present paper, we report three crystal structures of XylC in complex with Tris, xylobiose and xylose at 1.48-2.05 Å (1 Å=0.1 nm) resolution. XylC assembles into a tetramer, and each monomer comprises two distinct domains. The core domain is a right-handed parallel ß-helix (residues 1-75 and 201-638) and the flanking region (residues 76-200) folds into a ß-sandwich domain. The enzyme contains an open carbohydrate-binding cleft, allowing accommodation of longer xylo-oligosaccharides. On the basis of the crystal structures and in agreement with previous kinetic data, we propose that XylC cleaves the glycosidic bond by the retaining mechanism using two acidic residues Asp382 (nucleophile) and Glu405 (general acid/base). In addition to the active site, nine other xylose-binding sites were consistently observed in each of the four monomers, providing a possible reason for the high tolerance of product inhibition. [ABSTRACT FROM AUTHOR]

The endoglucanase EglA from Piromyces rhizinflata found in cattle stomach belongs to the GH5 family of glycoside hydrolases. The crystal structure of the catalytic domain of EglA shows the (β/α)8-barrel fold typical of GH5 enzymes. Adjacent to the active site of EglA, a loop containing a disulfide bond not found in other similar structures may participate in substrate binding. Because the active site was blocked by the N-terminal His tag of a neighbouring protein molecule in the crystal, enzyme-substrate complexes could not be obtained by soaking but were prepared by cocrystallization. The E154A mutant structure with a cellotriose bound to the −3, −2 and −1 subsites shows an extensive hydrogen-bonding network between the enzyme and the substrate, along with a stacking interaction between Trp44 and the −3 sugar. A possible dimer was observed in the crystal structure, but retention of activity in the E242A mutant suggested that the enzyme probably does not function as a dimer in solution. On the other hand, the first 100 amino acids encoded by the original cDNA fragment are very similar to those in the last third of the (β/α)8-barrel fold, indicating that EglA comprises at least two catalytic domains acting in tandem. [ABSTRACT FROM AUTHOR]

β-1,3-1,4-Glucanases catalyze the specific hydrolysis of internal β-1,4-glycosidic bonds adjacent to the 3- O-substituted glucose residues in mixed-linked β-glucans. The thermophilic glycoside hydrolase CtGlu16A from Clostridium thermocellum exhibits superior thermal profiles, high specific activity and broad pH adaptability. Here, the catalytic domain of CtGlu16A was expressed in Escherichia coli, purified and crystallized in the trigonal space group P3121, with unit-cell parameters a = b = 74.5, c = 182.9 Å, by the sitting-drop vapour-diffusion method and diffracted to 1.95 Å resolution. The crystal contains two protein molecules in an asymmetric unit. Further structural determination and refinement are in progress. [ABSTRACT FROM AUTHOR]

The β-L-arabinofuranosidase (HypBA1) from Bifidobacterium longum JCM 1217 hydrolyzes the β-1,2-linked arabinofuranose disaccharide to release L-arabinoses. HypBA1 was classified into glycoside hydrolase family 127 (GH127) by the CAZy website (). The enzyme was expressed in Escherichia coli and the purified recombinant protein was crystallized. Crystals belonging to the primitive hexagonal space group P3 x21, with unit-cell parameters a = b = 75.9, c = 254.0 Å, were obtained by the sitting-drop vapour-diffusion method and diffracted to 2.78 Å resolution. A BLASTP search () of the Protein Data Bank did not reveal any similar crystal structures. Structural determination by using SeMet MAD and MIR methods is in progress. [ABSTRACT FROM AUTHOR]

The glycoside hydrolase 10 (GH10) xylanase from Streptomyces sp. 9 (XynAS9) can operate in a broad range of pH and temperature, and thus is a potential candidate for commercial applications. Recently, we engineered XynAS9 via mutating several residues in accordance with the consensus sequences of GH10 thermophilic xylanases in an attempt to improve the enzyme thermostability and thermotolerance. The most promising effects were observed in the double mutant V81P/G82E. In order to investigate the molecular mechanism of the improved thermal profile of XynAS9, complex crystal structures of the wild type (WT) and mutant (MT) enzyme were solved at 1.88–2.05 Å resolution. The structures reveal a classical GH10 (β/α) 8 TIM-barrel fold. In MT XynAS9, E82 forms several interactions to its neighboring residues, which might aid in stabilizing the local structure. Furthermore, the MT structure showed lower B factors for individual residues compared to the WT structure, reflecting the increased MT protein rigidity. Analyses of the XynAS9 structures also delineate the detailed enzyme–substrate interaction network. More importantly, possible explanations for the enhanced thermal profiles of MT XynAS9 are proposed, which may be a useful strategy for enzyme engineering in the future. [ABSTRACT FROM AUTHOR]