No abstract

NkBgl, a β-glucosidase from Neotermes koshunensis, is a β-retaining glycosyl hydrolase family 1 enzyme that cleaves β-glucosidic linkages in disaccharide or glucose-substituted molecules. β-Glucosidases have been widely used in several applications. For example, mutagenesis of the attacking nucleophile in β-glucosidase has been conducted to convert it into a glycosynthase for the synthesis of oligosaccharides. Here, several high-resolution structures of wild-type or mutated NkBgl in complex with different ligand molecules are reported. In the wild-type NkBgl structures it was found that glucose-like glucosidase inhibitors bind to the glycone-binding pocket, allowing the buffer molecule HEPES to remain in the aglycone-binding pocket. In the crystal structures of NkBgl E193A, E193S and E193D mutants Glu193 not only acts as the catalytic acid/base but also plays an important role in controlling substrate entry and product release. Furthermore, in crystal structures of the NkBgl E193D mutant it was found that new glucoconjugates were generated by the conjugation of glucose (hydrolyzed product) and HEPES/EPPS/opipramol (buffer components). Based on the wild-type and E193D-mutant structures of NkBgl, the glucosidic bond of cellobiose or salicin was hydrolyzed and a new bond was subsequently formed between glucose and HEPES/EPPS/opipramol to generate new glucopyranosidic products through the transglycosylation reaction in the NkBgl E193D mutant. This finding highlights an innovative way to further improve β-glucosidases for the enzymatic synthesis of oligosaccharides.

Laminarinases hydrolyzing the ␤-1,3-linkage of glucans play essential roles in microbial saccharide degradation. Here we report the crystal structures at 1.65-1.82 Å resolution of the catalytic domain of laminarinase from the thermophile Thermotoga maritima with various space groups in the ligand-free form or in the presence of inhibitors gluconolactone and cetyltrimethylammonium. Ligands were bound at the cleft of the active site near an enclosure formed by Trp-232 and a flexible GASIG loop. A closed configuration at the active site cleft was observed in some molecules. The loop flexibility in the enzyme may contribute to the regulation of endo-or exo-activity of the enzyme and a preference to release laminaritrioses in long chain carbohydrate hydrolysis. Glu-137 and Glu-132 are proposed to serve as the proton donor and nucleophile, respectively, in the retaining catalysis of hydrolyzation. Calcium ions in the crystallization media are found to accelerate crystal growth. Comparison of laminarinase and endoglucanase structures revealed the subtle difference of key residues in the active site for the selection of ␤-1,3-glucan and ␤-1,4-glucan substrates, respectively. Arg-85 may be pivotal to ␤-1,3-glucan substrate selection. The similarity of the structures between the laminarinase catalytic domain and its carbohydrate-binding modules may have evolutionary relevance because of the similarities in their folds.Thermotoga maritima is a hyperthermophilic, anaerobic, and fermentive saccharolytic bacterium, catabolizing sugars and its polymers to make energy. Laminarinase (3-␤-D-glucan glucanohydrolase; EC 3.2.1.39, Lam), 3 an endoglucosidase, hydrolyzes internal ␤-1,3-glucosyl linkages in ␤-D-glucans and is therefore crucial in carbohydrate degradation for nutrient uptake and energy production in bacteria. According to the sequenced genome of T. maritima MSB8 (1), the laminarinase gene encodes the enzyme composed of a catalytic domain and two carbohydrate-binding modules (CBMs) connected by a linker region on each terminus. The structure of TmLamCBM2, located on the C terminus, has previously been determined by Boraston et al. (2). The coexistence of CBM with catalytic domains is widespread in many modular bacterial polysaccharide hydrolases that contain separately folding modules (3). The prevalent role of CBMs is to facilitate the association of substrates with the catalytic module; moreover, they sometime boost the reaction efficiency of the catalytic domain (4, 5). The modularity in biological macromolecules draws scientists' attention in biocatalyst designs (6). Because of the substrate diversity among glycosyl hydrolases (GHs), it is not easy to classify these enzymes according to their substrate specificity. Henrissat and Bairoch (7) developed a sequence similarity-based classification to categorize GH enzymes as an alternative to the traditional enzyme classification system. Except for a laminaripentaose-producing ␤-1,3-glucanase from Streptomyces matensis, which belongs to GH-64 (8), most of the bacterial la...