Glucosyltransferase TeGSS from Thermosynechococcus elongatus produces an α-1,2-glucan.

Here, we enzymatically produced a novel α-1,2-glucan, glucosylsucrose, that has a chemical structure significantly different from that of other glucans. This structural difference suggests its potential to modulate new physiological activities compared to known glucans. The enzyme TeGSS catalyzes the synthesis of this α-1,2-glucan from sucrose and UDP-glucose (UDPG). Using NMR spectroscopy, we elucidated the chemical structures of TeGSS-synthesized glucosylsucrose tri-, tetra-, and penta-saccharides in which the monosaccharide units are linked by α-1,2-glycosidic bonds. We also report the crystal structures of TeGSS co-crystallized with UDP and glucosylsucrose tri- and tetra-saccharides. Site-directed mutagenesis of residues in and around the TeGSS catalytic center has allowed us to propose a concerted SNi mechanism of action. Finally, we developed an enzyme-coupled reaction involving TeGSS and SuSyAc that allows production of UDPG for the synthesis of α-1,2-glucan.

Linear ß-1,2-glucans trigger immune hallmarks and enhance disease resistance in plants.

Immune responses in plants are triggered by molecular patterns or elicitors, recognized by plant pattern recognition receptors. Such molecular patterns are consequence of host-pathogen interactions and the response cascade activated after their perception is known as pattern-triggered immunity (PTI). Glucans have emerged as key players in PTI, but the ability of certain glucans to stimulate defensive responses in plants remains understudied. This work focused on identifying novel glucan oligosaccharides as molecular patterns. The ability of various microorganism-derived glucans to prompt PTI responses was tested, revealing that specific microbial-derived molecules, such as short linear ß-1,2-glucans, trigger this response in plants by increasing the production of reactive oxygen species (ROS), MAP kinase phosphorylation, and differential expression of defence-related genes in Arabidopsis thaliana. Pretreatments with ß-1,2-glucan trisaccharide (B2G3) improved Arabidopsis defence against bacterial and fungal infections in a hypersusceptible genotype. The knowledge generated was then transferred to the monocotyledonous model species maize and wheat, confirming that these plants also respond to ß-1,2-glucans, with increased ROS production and improved protection against fungal infections following B2G3 pretreatments. In summary, as with other ß-glucans, plants perceive ß-1,2-glucans as warning signals and stimulate defence responses against phytopathogens.

Functional and structural analysis of a cyclization domain in a cyclic ß-1,2-glucan synthase.

Cyclic ß-1,2-glucan synthase (CGS) is a key enzyme in production of cyclic ß-1,2-glucans (CßGs) which are involved in bacterial infection or symbiosis to host organisms. Nevertheless, a mechanism of cyclization, the final step in the CGS reaction, has not been fully understood. Here we performed functional and structural analyses of the cyclization domain of CGS alone from Thermoanaerobacter italicus (TiCGSCy). We first found that ß-glucosidase-resistant compounds are produced by TiCGSCy with linear ß-1,2-glucans as substrates. The 1H-NMR analysis revealed that these products are CßGs. Next, action pattern analyses using ß-1,2-glucooligosaccharides revealed a unique reaction pattern: exclusive transglycosylation without hydrolysis and a hexasaccharide being the minimum length of the substrate. These analyses also showed that longer substrate ß-1,2-glucooligosaccharides are preferred, being consistent with the fact that CGSs generally produce CßGs with degrees of polymerization of around 20. Finally, the overall structure of the cyclization domain of TiCGSCy was found to be similar to those of ß-1,2-glucanases in phylogenetically different groups. Meanwhile, the identified catalytic residues indicated clear differences in the reaction pathways between these enzymes. Overall, we propose a novel reaction mechanism of TiCGSCy. Thus, the present group of CGSs defines a new glycoside hydrolase family, GH189. KEY POINTS: • It was clearly evidenced that cyclization domain alone produces cyclic ß-1,2-glucans. • The domain exclusively catalyzes transglycosylation without hydrolysis. • The present catalytic domain defines as a new glycoside hydrolase family 189.

Characterization and structural analyses of a novel glycosyltransferase acting on the ß-1,2-glucosidic linkages.

The IALB_1185 protein, which is encoded in the gene cluster for endo-ß-1,2-glucanase homologs in the genome of Ignavibacterium album, is a glycoside hydrolase family (GH) 35 protein. However, most known GH35 enzymes are ß-galactosidases, which is inconsistent with the components of this gene cluster. Thus, IALB_1185 is expected to possess novel enzymatic properties. Here, we showed using recombinant IALB_1185 that this protein has glycosyltransferase activity toward ß-1,2-glucooligosaccharides, and that the kinetic parameters for ß-1,2-glucooligosaccharides are not within the ranges for general GH enzymes. When various aryl- and alkyl-glucosides were used as acceptors, glycosyltransfer products derived from these acceptors were subsequently detected. Kinetic analysis further revealed that the enzyme has wide aglycone specificity regardless of the anomer, and that the ß-1,2-linked glucose dimer sophorose is an appropriate donor. In the complex of wild-type IALB_1185 with sophorose, the electron density of sophorose was clearly observed at subsites -1 and +1, whereas in the E343Q mutant-sophorose complex, the electron density of sophorose was clearly observed at subsites +1 and +2. This observation suggests that binding at subsites -1 and +2 competes through Glu102, which is consistent with the preference for sophorose as a donor and unsuitability of ß-1,2-glucooligosaccharides as acceptors. A pliable hydrophobic pocket that can accommodate various aglycone moieties was also observed in the complex structures with various glucosides. Overall, our biochemical and structural data are indicative of a novel enzymatic reaction. We propose that IALB_1185 be redefined ß-1,2-glucooligosaccharide:d-glucoside ß-d-glucosyltransferase as a systematic name and ß-1,2-glucosyltransferase as an accepted name.

Enzymatic control and evaluation of degrees of polymerization of ß-(1→2)-glucans.

ß-(1 â†’ 2)-Glucans can be synthesized by 1,2-ß-oligoglucan phosphorylase using ß-(1 â†’ 2)-glucooligosaccharides as acceptors and α-d-glucose 1-phosphate as a donor. Using phosphorolysis of sucrose as a source of α-d-glucose 1-phosphate, we generated ß-(1 â†’ 2)-glucans with degrees of polymerization (DPs) up to approximately 280. Average DPs up to approximately 1000 were obtained using ß-(1 â†’ 2)-glucan with average DP of 160 as an acceptor and pure α-d-glucose 1-phosphate as a donor. A colorimetric assay of the ß-glucosidase activity against the ß-(1 â†’ 2)-glucan products was used to determine their DPs.

Cyclic ß-(1, 2)-glucan blended poly DL lactic co glycolic acid (PLGA 10:90) nanoparticles for drug delivery.

Our group had previously reported the encapsulation efficiency of cyclic ß-(1, 2)-glucan for various drugs. The current study is aimed at evaluating the use of glucan as a drug carrier system by blending with poly lactic-co- glycolic acid (L:G = 10:90). Nanoparticles of glucan (0.5, 5, 10 and 20 wt %) blended with PLGA and gentamicin were synthesized. Encapsulation efficiency was higher for the blends (93% with 20 wt % of glucan) than the PLGA alone (79.8%). The presence of glucan enhanced both the biodegradability, and biocompatibility of PLGA. Degradation of the nanoparticles in vitro, was autocatalytic with an initial burst release of active drug and the release profile was modeled using the Korsmeyer-Peppas scheme. In vivo studies indicated that the drug released from the blends had high volume of distribution, and greater clearance from the system. Pharmacokinetics of the drug was predicted using a double exponential decay model. Blending with PLGA improved the drug release characteristics of the cyclic ß-(1, 2)-glucan.

Large-scale preparation of ß-1,2-glucan using quite a small amount of sophorose.

A large amount of ß-1,2-glucan was produced enzymatically from quite a small amount of sophorose as an acceptor material through three synthesis steps using a sucrose phosphorylase and a 1,2-ß-oligoglucan phosphorylase. The first synthesis step was performed in a 200 µL of a reaction solution containing 5 mM sophorose and 1.0 M sucrose. ß-1,2-Glucan in a part of the resultant solution was hydrolyzed to ß-1,2-glucooligosaccharides by a ß-1,2-glucanase. The second synthesis was performed in 25 times the volume for the first synthesis. The hydrolysate solution (1% volume of the reaction solution) was used as an acceptor. After treatment with the ß-1,2-glucanase again, the third synthesis was performed 200 times the volume for the second synthesis (1 L). The reaction yield of ß-1,2-glucan at each synthesis was 93%, 76% and 91%. Finally, more than 140 g of ß-1,2-glucan was synthesized using approximately 20 µg of sophorose as the starting acceptor material. Abbreviations: DPs: degrees of polymerization; SOGP: 1,2-ß-oligoglucan phosphorylase; Sopns: ß-1,2-glucooligosaccharides with DP of n; Glc1P: α-glucose 1-phosphate; SucP: sucrose phosphorylase from Bifidobacterium longum subsp. longum; SGL: ß-1,2-glucanase; CaSGL: Chy400_4174 protein; TLC: thin layer chromatography; GOPOD: glucose oxidase/peroxidase; PGM: phosphoglucomutase; G6PDH: glucose 6-phosphate dehydrogenase.

Identification, characterization, and structural analyses of a fungal endo-ß-1,2-glucanase reveal a new glycoside hydrolase family.

endo-ß-1,2-Glucanase (SGL) is an enzyme that hydrolyzes ß-1,2-glucans, which play important physiological roles in some bacteria as a cyclic form. To date, no eukaryotic SGL has been identified. We purified an SGL from Talaromyces funiculosus (TfSGL), a soil fungus, to homogeneity and then cloned the complementary DNA encoding the enzyme. TfSGL shows no significant sequence similarity to any known glycoside hydrolase (GH) families, but shows significant similarity to certain eukaryotic proteins with unknown functions. The recombinant TfSGL (TfSGLr) specifically hydrolyzed linear and cyclic ß-1,2-glucans to sophorose (Glc-ß-1,2-Glc) as a main product. TfSGLr hydrolyzed reducing-end-modified ß-1,2-gluco-oligosaccharides to release a sophoroside with the modified moiety. These results indicate that TfSGL is an endo-type enzyme that preferably releases sophorose from the reducing end of substrates. Stereochemical analysis demonstrated that TfSGL is an inverting enzyme. The overall structure of TfSGLr includes an (α/α)6 toroid fold. The substrate-binding mode was revealed by the structure of a Michaelis complex of an inactive TfSGLr mutant with a ß-1,2-glucoheptasaccharide. Mutational analysis and action pattern analysis of ß-1,2-gluco-oligosaccharide derivatives revealed an unprecedented catalytic mechanism for substrate hydrolysis. Glu-262 (general acid) indirectly protonates the anomeric oxygen at subsite -1 via the 3-hydroxy group of the Glc moiety at subsite +2, and Asp-446 (general base) activates the nucleophilic water via another water. TfSGLr is apparently different from a GH144 SGL in the reaction and substrate recognition mechanism based on structural comparison. Overall, we propose that TfSGL and closely-related enzymes can be classified into a new family, GH162.

Colorimetric determination of ß-1,2-glucooligosaccharides for an enzymatic assay using 3-methyl-2-benzothiazolinonehydrazone.

A colorimetric determination method measuring the reducing ends of sugars is usually used for quantitative evaluation of polysaccharide-degrading activity of endo-type enzymes. However, no appropriate colorimetric method has been established for enzymatic assay of ß-1,2-glucanases, which produce ß-1,2-glucooligosaccharides from ß-1,2-glucans. The Anthon-MBTH method has been potentially the most adaptable for color development of ß-1,2-glucooligosaccharides among various known colorimetric methods for detecting the reducing power of oligosaccharides, since the difference between sophorose and other ß-1,2-glucooligosaccharides in absorbance is relatively small. Almost the same color development was obtained for ß-1,2-glucooligosaccharides when the heating time with the Anthon-MBTH method was changed. The kind of base and concentration of dithiothreitol did not markedly affect the color development. Most buffer components, salts and a chelating reagent used for usual enzymatic experiments did not inhibit color development. Furthermore, assay was performed successfully for a ß-1,2-glucanase using the modified MBTH method.

Structural and thermodynamic insights into ß-1,2-glucooligosaccharide capture by a solute-binding protein in Listeria innocua.

ß-1,2-Glucans are bacterial carbohydrates that exist in cyclic or linear forms and play an important role in infections and symbioses involving Gram-negative bacteria. Although several ß-1,2-glucan-associated enzymes have been characterized, little is known about how ß-1,2-glucan and its shorter oligosaccharides (Sop n s) are captured and imported into the bacterial cell. Here, we report the biochemical and structural characteristics of the Sop n -binding protein (SO-BP, Lin1841) associated with the ATP-binding cassette (ABC) transporter from the Gram-positive bacterium Listeria innocua Calorimetric analysis revealed that SO-BP specifically binds to Sop n s with a degree of polymerization of 3 or more, with Kd values in the micromolar range. The crystal structures of SO-BP in an unliganded open form and in closed complexes with tri-, tetra-, and pentaoligosaccharides (Sop3-5) were determined to a maximum resolution of 1.6 Å. The binding site displayed shape complementarity to Sop n , which adopted a zigzag conformation. We noted that water-mediated hydrogen bonds and stacking interactions play a pivotal role in the recognition of Sop3-5 by SO-BP, consistent with its binding thermodynamics. Computational free-energy calculations and a mutational analysis confirmed that interactions with the third glucose moiety of Sop n s are significantly responsible for ligand binding. A reduction in unfavorable changes in binding entropy that were in proportion to the lengths of the Sop n s was explained by conformational entropy changes. Phylogenetic and sequence analyses indicated that SO-BP ABC transporter homologs, glycoside hydrolases, and other related proteins are co-localized in the genomes of several bacteria. This study may improve our understanding of bacterial ß-1,2-glucan metabolism and promote the discovery of unidentified ß-1,2-glucan-associated proteins.

Function and structure relationships of a ß-1,2-glucooligosaccharide-degrading ß-glucosidase.

BT_3567 protein, a putative ß-glucosidase from Bacteroides thetaiotaomicron, exhibits higher activity toward Sop3-5 (Sopn , n: degree of polymerization of ß-1,2-glucooligosaccharides) than toward Sop2 , unlike a known ß-glucosidase from Listeria innocua which predominantly prefers Sop2 . In the complex structure determined by soaking of a D286N mutant crystal with Sop4 , a Sop3 moiety was observed at subsites -1 to +2. The glucose moiety at subsite +2 forms a hydrogen bond with Asn81, which is replaced with Gly in the L. innocua ß-glucosidase. The Km values of the N81G mutant for Sop3-5 are much higher than those of the wild-type, suggesting that Asn81 contributes to the binding to substrates longer than Sop3 .

Biochemical and structural analyses of a bacterial endo-ß-1,2-glucanase reveal a new glycoside hydrolase family.

ß-1,2-Glucan is an extracellular cyclic or linear polysaccharide from Gram-negative bacteria, with important roles in infection and symbiosis. Despite ß-1,2-glucan's importance in bacterial persistence and pathogenesis, only a few reports exist on enzymes acting on both cyclic and linear ß-1,2-glucan. To this end, we purified an endo-ß-1,2-glucanase to homogeneity from cell extracts of the environmental species Chitinophaga arvensicola, and an endo-ß-1,2-glucanase candidate gene (Cpin_6279) was cloned from the related species Chitinophaga pinensis The Cpin_6279 protein specifically hydrolyzed linear ß-1,2-glucan with polymerization degrees of ≥5 and a cyclic counterpart, indicating that Cpin_6279 is an endo-ß-1,2-glucananase. Stereochemical analysis demonstrated that the Cpin_6279-catalyzed reaction proceeds via an inverting mechanism. Cpin_6279 exhibited no significant sequence similarity with known glycoside hydrolases (GHs), and thus the enzyme defines a novel GH family, GH144. The crystal structures of the ligand-free and complex forms of Cpin_6279 with glucose (Glc) and sophorotriose (Glc-ß-1,2-Glc-ß-1,2-Glc) determined up to 1.7 Å revealed that it has a large cavity appropriate for polysaccharide degradation and adopts an (α/α)6-fold slightly similar to that of GH family 15 and 8 enzymes. Mutational analysis indicated that some of the highly conserved acidic residues in the active site are important for catalysis, and the Cpin_6279 active-site architecture provided insights into the substrate recognition by the enzyme. The biochemical characterization and crystal structure of this novel GH may enable discovery of other ß-1,2-glucanases and represent a critical advance toward elucidating structure-function relationships of GH enzymes.

Generation and characterization of ß1,2-gluco-oligosaccharide probes from Brucella abortus cyclic ß-glucan and their recognition by C-type lectins of the immune system.

The ß1,2-glucans produced by bacteria are important in invasion, survival and immunomodulation in infected hosts be they mammals or plants. However, there has been a lack of information on proteins which recognize these molecules. This is partly due to the extremely limited availability of the sequence-defined oligosaccharides and derived probes for use in the study of their interactions. Here we have used the cyclic ß1,2-glucan (CßG) of the bacterial pathogen Brucella abortus, after removal of succinyl side chains, to prepare linearized oligosaccharides which were used to generate microarrays. We describe optimized conditions for partial depolymerization of the cyclic glucan by acid hydrolysis and conversion of the ß1,2-gluco-oligosaccharides, with degrees of polymerization 2-13, to neoglycolipids for the purpose of generating microarrays. By microarray analyses, we show that the C-type lectin receptor DC-SIGNR, like the closely related DC-SIGN we investigated earlier, binds to the ß1,2-gluco-oligosaccharides, as does the soluble immune effector serum mannose-binding protein. Exploratory studies with DC-SIGN are suggestive of the recognition also of the intact CßG by this receptor. These findings open the way to unravelling mechanisms of immunomodulation mediated by ß1,2-glucans in mammalian systems.

Modulation of acceptor specificity of Ruminococcus albus cellobiose phosphorylase through site-directed mutagenesis.

Cellobiose phosphorylase (EC 2.4.1.20, CBP) catalyzes the reversible phosphorolysis of cellobiose to α-D-glucose 1-phosphate (Glc1P) and d-glucose. Cys485, Tyr648, and Glu653 of CBP from Ruminococcus albus, situated at the +1 subsite, were mutated to modulate acceptor specificity. C485A, Y648F, and Y648V were active enough for analysis. Their acceptor specificities were compared with the wild type based on the apparent kinetic parameters determined in the presence of 10 mM Glc1P. C485A showed higher preference for D-glucosamine than the wild type. Apparent kcat/Km values of Y648F for D-mannose and 2-deoxy-D-glucose were 8.2- and 4.0-fold higher than those of the wild type, respectively. Y648V had synthetic activity toward N-acetyl-D-glucosamine, while the other variants did not. The oligosaccharide production in the presence of the same concentrations of wild type and each mutant was compared. C485A produced 4-O-ß-D-glucopyranosyl-D-glucosamine from 10 mM Glc1P and D-glucosamine at a rate similar to the wild type. Y648F and Y648V produced 4-O-ß-D-glucopyranosyl-D-mannose and 4-O-ß-D-glucopyranosyl-N-acetyl-D-glucosamine much more rapidly than the wild type when D-mannose and N-acetyl-D-glucosamine were used as acceptors, respectively. After a 4h reaction, the amounts of 4-O-ß-D-glucopyranosyl-D-mannose and 4-O-ß-D-glucopyranosyl-N-acetyl-D-glucosamine produced by Y648F and Y648V were 5.9- and 12-fold higher than the wild type, respectively.