Oligomeric Structure of Yeast and Other Invertases Governs Specificity.

Invertases, or ß-fructofuranosidases, are metabolic enzymes widely distributed among plants and microorganisms that hydrolyze sucrose and release fructose from various substrates. Invertase was one of the earliest discovered enzymes, first investigated in the mid-nineteenth century, becoming a classical model used in the primary biochemical studies on protein synthesis, activity, and the secretion of glycoproteins. However, it was not until 20 years ago that a member of this family of enzymes was structurally characterized, showing a bimodular arrangement with a ß-propeller catalytic domain, and a ß-sandwich domain with unknown function. Since then, many studies on related plant and fungal enzymes have revealed them as basically monomeric. By contrast, all yeast enzymes in this family that have been characterized so far have shown sophisticated oligomeric structures mediated by the non-catalytic domain, which is also involved in substrate binding, and how this assembly determines the particular specificity of each enzyme. In this chapter, we will review the available structures of yeast invertases to elucidate the mechanism regulating oligomer formation and compare them with other reported dimeric invertases in which the oligomeric assembly has no apparent functional implications. In addition, recent work on a new family of invertases with absolute specificity for the α-(1,2)-bond of sucrose found in cyanobacteria and plant invertases is highlighted.

Multifaceted roles of plant glycosyl hydrolases during pathogen infections: more to discover.

MAIN CONCLUSION: Carbohydrates are hydrolyzed by a family of carbohydrate-active enzymes (CAZymes) called glycosidases or glycosyl hydrolases. Here, we have summarized the roles of various plant defense glycosidases that possess different substrate specificities. We have also highlighted the open questions in this research field. Glycosidases or glycosyl hydrolases (GHs) are a family of carbohydrate-active enzymes (CAZymes) that hydrolyze glycosidic bonds in carbohydrates and glycoconjugates. Compared to those of all other sequenced organisms, plant genomes contain a remarkable diversity of glycosidases. Plant glycosidases exhibit activities on various substrates and have been shown to play important roles during pathogen infections. Plant glycosidases from different GH families have been shown to act upon pathogen components, host cell walls, host apoplastic sugars, host secondary metabolites, and host N-glycans to mediate immunity against invading pathogens. We could classify the activities of these plant defense GHs under eleven different mechanisms through which they operate during pathogen infections. Here, we have provided comprehensive information on the catalytic activities, GH family classification, subcellular localization, domain structure, functional roles, and microbial strategies to regulate the activities of defense-related plant GHs. We have also emphasized the research gaps and potential investigations needed to advance this topic of research.

Synthesis and Structural Investigation of Foldamer-Based Iodine-Supported Artificial Glycosidases.

Glycosidases are a type of enzyme that hydrolytically cleave carbohydrates and form glycans for biologically important processes. The inadequacies of glycosidases or their genetic abnormalities are responsible for various diseases. Thus, the development of glycosidase mimetics is of great importance. We have designed and synthesized an enzyme mimetic containing l-phenylalanine, α-aminoisobutyric acid (Aib), l-leucine, and m-Nifedipine. From X-ray crystallography, the foldamer adopts a ß-hairpin conformation stabilized by two 10-member and one 18-member NH⋅⋅⋅O=C hydrogen bonds. Moreover, the foldamer was found to be highly efficient in hydrolysing ethers and glycosides in the presence of iodine at room temperature. Further, X-ray analysis shows the backbone conformation of the enzyme mimetic to be almost unchanged after the glycosidase reaction. This is the first example of iodine-supported artificial glycosidase activity with an enzyme mimic at ambient conditions.

Control of Substrate Conformation by Hydrogen Bonding in a Retaining ß-Endoglycosidase.

Bacterial ß-glycosidases are hydrolytic enzymes that depolymerize polysaccharides such as ß-cellulose, ß-glucans and ß-xylans from different sources, offering diverse biomedical and industrial uses. It has been shown that a conformational change of the substrate, from a relaxed 4 C1 conformation to a distorted 1 S3 /1,4 B conformation of the reactive sugar, is necessary for catalysis. However, the molecular determinants that stabilize the substrate's distortion are poorly understood. Here we use quantum mechanics/molecular mechanics (QM/MM)-based molecular dynamics methods to assess the impact of the interaction between the reactive sugar, i. e. the one at subsite -1, and the catalytic nucleophile (a glutamate) on substrate conformation. We show that the hydrogen bond involving the C2 exocyclic group and the nucleophile controls substrate conformation: its presence preserves sugar distortion, whereas its absence (e.g. in an enzyme mutant) knocks it out. We also show that 2-deoxy-2-fluoro derivatives, widely used to trap the reaction intermediates by X-ray crystallography, reproduce the conformation of the hydrolysable substrate at the experimental conditions. These results highlight the importance of the 2-OH⋅⋅⋅nucleophile interaction in substrate recognition and catalysis in endo-glycosidases and can inform mutational campaigns aimed to search for more efficient enzymes.

Selective Wine Aroma Enhancement through Enzyme Hydrolysis of Glycosidic Precursors.

Selective enhancement of wine aroma was achieved using a broad spectrum of exogenous glycosidases. Eight different enzyme preparations were added to Verdejo wine, resulting in an increase in the levels of varietal volatile compounds compared to the control wine after 15 days of treatment. The enzyme preparations studied were robust under winemaking conditions (sulfur dioxide, reducing sugars, and alcohol content), and no inhibition of ß-glucosidase activity was observed. Significant differences were detected in four individual terpenes (α-terpineol, terpinen-4-ol, α-pinene, and citronellal) and benzyl alcohol in all the treated wines compared to the control wine, contributing to the final wine to varying degrees. In addition, a significant increase in the other aromatic compounds was observed, which showed different patterns depending on the enzyme preparation that was tested. The principal component analysis of the data revealed the possibility of modulating the different aromatic profiles of the final wines depending on the enzyme preparation used. Taking these results into account, enhancement of the floral, balsamic, and/or fruity notes of wines is possible by using a suitable commercial enzyme preparation.

How microbial glycosyl hydrolase activity in the gut mucosa initiates microbial cross-feeding.

The intestinal epithelium is protected from direct contact with gut microbes by a mucus layer. This mucus layer consists of secreted mucin glycoproteins. The outer mucus layer in the large intestine forms a niche that attracts specific gut microbiota members of which several gut commensals can degrade mucin. Mucin glycan degradation is a complex process that requires a broad range of glycan degrading enzymes, as mucin glycans are intricate and diverse molecules. Consequently, it is hypothesized that microbial mucin breakdown requires concerted action of various enzymes in a network of multiple resident microbes in the gut mucosa. This review investigates the evolutionary relationships of microbial carbohydrate-active enzymes that are potentially involved in mucin glycan degradation and focuses on the role that microbial enzymes play in the degradation of gut mucin glycans in microbial cross-feeding and syntrophic interactions.

Tuning the Transglycosylation Reaction of a GH11 Xylanase by a Delicate Enhancement of its Thumb Flexibility.

Glycoside hydrolases (GHs) are attractive tools for multiple biotechnological applications. In conjunction with their hydrolytic function, GHs can perform transglycosylation under specific conditions. In nature, oligosaccharide synthesis is performed by glycosyltransferases (GTs); however, the industrial use of GTs is limited by their instability in solution. A key difference between GTs and GHs is the flexibility of their binding site architecture. We have used the xylanase from Bacillus circulans (BCX) to study the interplay between active-site flexibility and transglycosylation. Residues of the BCX "thumb" were substituted to increase the flexibility of the enzyme binding site. Replacement of the highly conserved residue P116 with glycine shifted the balance of the BCX enzymatic reaction toward transglycosylation. The effects of this point mutation on the structure and dynamics of BCX were investigated by NMR spectroscopy. The P116G mutation induces subtle changes in the configuration of the thumb and enhances the millisecond dynamics of the active site. Based on our findings, we propose the remodelling of the GH enzymes glycon site flexibility as a strategy to improve the transglycosylation efficiency of these biotechnologically important catalysts.

Leaf Apoplast of Field-Grown Potato Analyzed by Quantitative Proteomics and Activity-Based Protein Profiling.

Multiple biotic and abiotic stresses challenge plants growing in agricultural fields. Most molecular studies have aimed to understand plant responses to challenges under controlled conditions. However, studies on field-grown plants are scarce, limiting application of the findings in agricultural conditions. In this study, we investigated the composition of apoplastic proteomes of potato cultivar Bintje grown under field conditions, i.e., two field sites in June-August across two years and fungicide treated and untreated, using quantitative proteomics, as well as its activity using activity-based protein profiling (ABPP). Samples were clustered and some proteins showed significant intensity and activity differences, based on their field site and sampling time (June-August), indicating differential regulation of certain proteins in response to environmental or developmental factors. Peroxidases, class II chitinases, pectinesterases, and osmotins were among the proteins more abundant later in the growing season (July-August) as compared to early in the season (June). We did not detect significant differences between fungicide Shirlan treated and untreated field samples in two growing seasons. Using ABPP, we showed differential activity of serine hydrolases and ß-glycosidases under greenhouse and field conditions and across a growing season. Furthermore, the activity of serine hydrolases and ß-glycosidases, including proteins related to biotic stress tolerance, decreased as the season progressed. The generated proteomics data would facilitate further studies aiming at understanding mechanisms of molecular plant physiology in agricultural fields and help applying effective strategies to mitigate biotic and abiotic stresses.

Secretome Analysis of Arabidopsis-Trichoderma atroviride Interaction Unveils New Roles for the Plant Glutamate:Glyoxylate Aminotransferase GGAT1 in Plant Growth Induced by the Fungus and Resistance against Botrytis cinerea.

The establishment of plant-fungus mutualistic interaction requires bidirectional molecular crosstalk. Therefore, the analysis of the interacting organisms secretomes would help to understand how such relationships are established. Here, a gel-free shotgun proteomics approach was used to identify the secreted proteins of the plant Arabidopsis thaliana and the mutualistic fungus Trichoderma atroviride during their interaction. A total of 126 proteins of Arabidopsis and 1027 of T. atroviride were identified. Among them, 118 and 780 were differentially modulated, respectively. Bioinformatic analysis unveiled that both organisms' secretomes were enriched with enzymes. In T. atroviride, glycosidases, aspartic endopeptidases, and dehydrogenases increased in response to Arabidopsis. Additionally, amidases, protein-serine/threonine kinases, and hydro-lyases showed decreased levels. Furthermore, peroxidases, cysteine endopeptidases, and enzymes related to the catabolism of secondary metabolites increased in the plant secretome. In contrast, pathogenesis-related proteins and protease inhibitors decreased in response to the fungus. Notably, the glutamate:glyoxylate aminotransferase GGAT1 was secreted by Arabidopsis during its interaction with T. atroviride. Our study showed that GGAT1 is partially required for plant growth stimulation and on the induction of the plant systemic resistance by T. atroviride. Additionally, GGAT1 seems to participate in the negative regulation of the plant systemic resistance against B. cinerea through a mechanism involving H2O2 production.

Sclerotium rolfsii lectin induces opposite effects on normal PBMCs and leukemic Molt-4 cells by recognising TF antigen and its variants as receptors.

Sclerotium rolfsii lectin (SRL) exerts apoptotic effect against various cancer cells and an antitumor activity on mice with colon and breast cancer xenografts. The current study aimed to explore its exquisite carbohydrate specificity on human peripheral blood mononuclear cells (PBMCs) and leukemic T-cells. SRL, showed strong binding (>98%) to resting/activated PBMCs, leukemic Molt-4 and Jurkat cell lines. The glycans mediated binding to these cells was effectively blocked by mucin and fetuin, exhibiting 97% and 94% inhibition respectively. SRL showed mitogenic stimulation of PBMCs at 10 µg/ml as determined by thymidine incorporation assay. In contrast, lectin induced a dose dependent growth inhibition of Molt-4 cells with 58% inhibition at 25 µg/ml. Many common membrane receptors in activated PBMCs, Molt 4 and Jurkat cells were identified by lectin blotting. However, membrane receptors that are recognized by SRL in normal resting PBMCs were totally different and are high molecular weight glycoproteins. Treatment of membrane receptors with glycosidases prior to lectin probing, revealed that fucosylated Thomsen-Friedenreich(TF) antigen glycans are increasingly expressed on transformed Molt-4 leukemic cells compared to other cells. The findings highlight the opposite effects of SRL on transformed and normal hematopoietic cells by recognizing different glycan-receptors. SRL has promising potential for diagnostics and therapeutic applications in leukaemia.

Cooperated biotransformation of ginsenoside extracts into ginsenoside 20(S)-Rg3 by three thermostable glycosidases.

AIMS: The aim of this work was to transform ginsenoside extract into the pharmacologically active minor ginsenoside 20(S)-Rg3 by three thermostable glycosidases. METHODS AND RESULTS: The GH1 thermostable beta-glucosidase Tpebgl1 from Thermotoga petrophlia was found to have the ability to convert ginsenosides Rb1 and Rb2. Its properties concerning ginsenoside conversion were systematically investigated. It had high specific activity on pNPG (162·20 U mg-1 ) and pNPArp (22·14 U mg-1 ). The Km and Vmax of Tpebgl1 for pNPG were 0·28 mmol l-1 and 470·2 U mg-1 and for pNPArp were 17·30 mmol l-1 and 74·28 U mg-1 . Therefore, it could successfully convert ginsenosides Rb1 and Rb2 into ginsenoside Rd, which has been proven by experiments in this paper then. Tpebgl1 also had good tolerance to glucose and some organic solvents. These made Tpebgl1 a good catalyst candidate for industrial application. Finally, it was applied to convert ginsenoside extract into the pharmacologically active minor ginsenoside 20(S)-Rg3, combined with thermostable ginsenoside Rc converting α-1,6-l-arabinofranosidase Tt-Afs and ginsenoside Rd converting ß-glucosidase Tpebgl3. A quantity of 10 g l-1 of ginsenoside extract was transformed into 3·93 g l-1 of Rg3 at 90°C, pH 5·0 for 3 h, with a corresponding molar conversion of 98·19%. CONCLUSION: The thermostable enzyme Tpebgl1 was found to be a ginsenoside-converting enzyme and successfully applied in the preparation of ginsenoside 20(S)-Rg3 from ginsenoside extract. The three-step cooperate  transformation system of ginsenoside extract was established by using Tpebgl1, Tt-Afs (a thermostable ginsenoside Rc converting α-1,6-l-arabinofranosidase) and Tpebgl3 (a thermostable ginsenoside Rb1 converting ß-glucosidase). SIGNIFICANCE AND IMPACT OF THE STUDY: Converting all the major ginsenosides into protopanaxadiol-type ginsenoside extract would greatly reduce the cost of ginsenoside Rg3 preparation. Enzymes from thermophilic bacteria can meet the requirement of higher reaction temperatures in industrial reactions for substrate solubility promotion and bacterial contamination prevention.

Activity of selected glycosidases and availability of their substrates in bovine placenta during pregnancy and parturition with and without retained foetal membranes.

The activity of glycosidases is crucial for the function and biological activity of proteins conjugated with sugar moieties, which play an important role in adhesion of cells during attachment and detachment of the foetal membranes. The aim of study was to describe the ability of bovine placental tissues to break down O-glycosidic bonds in different glycoproteins by the determination of activity of ß-galactosidase, α-l-fucosidase, ß-N-acetyl-hexosaminidase and sialidase in early-mid-pregnancy as well as at parturition with released and retained foetal membranes. Moreover, the availability of substrates for these glycosidases in placental homogenates was evaluated. Placental samples were collected from pregnant (2-4 months) cows in slaughterhouse (n = 8) as well as during Caesarean section and divided into released foetal membranes (n = 8) and retained foetal membranes (n = 8). Tissue homogenates were subjected to spectrofluorimetric and spectrophotometric determinations of enzyme activities as well as electrophoretic separations. Enzyme activities expressed changes within examined time with significant (p < .05) differences between pregnancy and physiological parturition in ß-N-acetyl-hexosaminidase and α-l-fucosidase in foetal part of placenta while in maternal part only in the latter one. Decreasing tendency in enzyme activity was noticed in foetal part of retained samples in comparison with released ones with significant (p < .05) differences in α-l-fucosidase activity. The analysis of staining of sugar moieties attached to selected proteins depicted availability of sugar molecules in examined tissues, but their patterns differed between samples. In conclusion, sugar moieties in conjugated proteins express changes in the course of pregnancy which is reflected by the alterations in activities of placental glycosidases.

Gangliosides and Neuroblastomas.

The focus of this review is the ganglio-series of glycosphingolipids found in neuroblastoma (NB) and the myriad of unanswered questions associated with their possible role(s) in this cancer. NB is one of the more common solid malignancies of children. Five-year survival for those diagnosed with low risk NB is 90-95%, while that for children with high-risk NB is around 40-50%. Much of the survival rate reflects age of diagnosis with children under a year having a much better prognosis than those over two. Identification of expression of GD2 on the surface of most NB cells led to studies of the effectiveness and subsequent approval of anti-GD2 antibodies as a treatment modality. Despite much success, a subset of patients, possibly those whose tumors fail to express concentrations of gangliosides such as GD1b and GT1b found in tumors from patients with a good prognosis, have tumors refractory to treatment. These observations support discussion of what is known about control of ganglioside synthesis, and their actual functions in NB, as well as their possible relationship to treatment response.

Dynamics of Ligand Binding to a Rigid Glycosidase*.

The single-domain GH11 glycosidase from Bacillus circulans (BCX) is involved in the degradation of hemicellulose, which is one of the most abundant renewable biomaterials in nature. We demonstrate that BCX in solution undergoes minimal structural changes during turnover. NMR spectroscopy results show that the rigid protein matrix provides a frame for fast substrate binding in multiple conformations, accompanied by slow conversion, which is attributed to an enzyme-induced substrate distortion. A model is proposed in which the rigid enzyme takes advantage of substrate flexibility to induce a conformation that facilitates the acyl formation step of the hydrolysis reaction.

Structural and mechanistic analysis of a ß-glycoside phosphorylase identified by screening a metagenomic library.

Glycoside phosphorylases have considerable potential as catalysts for the assembly of useful glycans for products ranging from functional foods and prebiotics to novel materials. However, the substrate diversity of currently identified phosphorylases is relatively small, limiting their practical applications. To address this limitation, we developed a high-throughput screening approach using the activated substrate 2,4-dinitrophenyl ß-d-glucoside (DNPGlc) and inorganic phosphate for identifying glycoside phosphorylase activity and used it to screen a large insert metagenomic library. The initial screen, based on release of 2,4-dinitrophenyl from DNPGlc in the presence of phosphate, identified the gene bglP, encoding a retaining ß-glycoside phosphorylase from the CAZy GH3 family. Kinetic and mechanistic analysis of the gene product, BglP, confirmed a double displacement ping-pong mechanism involving a covalent glycosyl-enzyme intermediate. X-ray crystallographic analysis provided insights into the phosphate-binding mode and identified a key glutamine residue in the active site important for substrate recognition. Substituting this glutamine for a serine swapped the substrate specificity from glucoside to N-acetylglucosaminide. In summary, we present a high-throughput screening approach for identifying ß-glycoside phosphorylases, which was robust, simple to implement, and useful in identifying active clones within a metagenomics library. Implementation of this screen enabled discovery of a new glycoside phosphorylase class and has paved the way to devising simple ways in which enzyme specificity can be encoded and swapped, which has implications for biotechnological applications.

Synthetic and Crystallographic Insight into Exploiting sp2 Hybridization in the Development of α-l-Fucosidase Inhibitors.

The sugar fucose plays a myriad of roles in biological recognition. Enzymes hydrolyzing fucose from glycoconjugates, α-l-fucosidases, are important targets for inhibitor and probe development. Here we describe the synthesis and evaluation of novel α-l-fucosidase inhibitors, with X-ray crystallographic analysis using an α-l-fucosidase from Bacteroides thetaiotamicron helping to lay a foundation for future development of inhibitors for this important enzyme class.

Multivalent Thiosialosides and Their Synergistic Interaction with Pathogenic Sialidases.

Sialidases (SAs) hydrolyze sialyl residues from glycoconjugates of the eukaryotic cell surface and are virulence factors expressed by pathogenic bacteria, viruses, and parasites. The catalytic domains of SAs are often flanked with carbohydrate-binding module(s) previously shown to bind sialosides and to enhance enzymatic catalytic efficiency. Herein, non-hydrolyzable multivalent thiosialosides were designed as probes and inhibitors of V. cholerae, T. cruzi, and S. pneumoniae (NanA) sialidases. NanA was truncated from the catalytic and lectinic domains (NanA-L and NanA-C) to probe their respective roles upon interacting with sialylated surfaces and the synthetically designed di- and polymeric thiosialosides. The NanA-L domain was shown to fully drive NanA binding, improving affinity for the thiosialylated surface and compounds by more than two orders of magnitude. Importantly, each thiosialoside grafted onto the polymer was also shown to reduce NanA and NanA-C catalytic activity with efficiency that was 3000-fold higher than that of the monovalent thiosialoside reference. These results extend the concept of multivalency for designing potent bacterial and parasitic sialidase inhibitors.

Structural basis of the inhibition of GH1 ß-glucosidases by multivalent pyrrolidine iminosugars.

The synthesis of multivalent pyrrolidine iminosugars via CuAAC click reaction between different pyrrolidine-azide derivatives and tri- or hexavalent alkynyl scaffolds is reported. The new multimeric compounds, together with the monomeric reference, were evaluated as inhibitors against two homologous GH1 ß-glucosidases (BglA and BglB from Paenibacillus polymyxa). The multivalent inhibitors containing an aromatic moiety in the linker between the pyrrolidine and the scaffold inhibited the octameric BglA (µM range) but did not show affinity against the monomeric BglB, despite the similarity between the active site of both enzymes. A modest multivalent effect (rp/n = 12) was detected for the hexavalent inhibitor 12. Structural analysis of the complexes between the monomeric and the trimeric iminosugar inhibitors (4 and 10) and BglA showed the insertion of the inhibitors at the active site of BglA, confirming a competitive mode of inhibition as indicated by enzyme kinetics. Additionally, structural comparison of the BglA/4 complex with the reported BglB/2F-glucose complex illustrates the key determinants responsible for the inhibitory effect and explains the reasons of the inhibition of BglA and the no inhibition of BglB. Potential inhibition of other ß-glucosidases with therapeutic relevance is discussed under the light of these observations.

Direct Stereoselective Aziridination of Cyclohexenols with 3-Amino-2-(trifluoromethyl)quinazolin-4(3H)-one in the Synthesis of Cyclitol Aziridine Glycosidase Inhibitors.

Cyclophellitol aziridine and its configurational and functional isomers are powerful covalent inhibitors of retaining glycosidases, and find application in fundamental studies on glycosidases, amongst others in relation to inherited lysosomal storage disorders caused by glycosidase malfunctioning. Few direct and stereoselective aziridination methodologies are known for the synthesis of cyclophellitol aziridines. Herein, we present our studies on the scope of direct 3-amino-2-(trifluoromethyl)quinazolin-4(3H)-one-mediated aziridination on a variety of configurational and functional cyclohexenol isosters. We demonstrate that the aziridination can be directed by an allylic or homoallylic hydroxyl through H-bonding and that steric hindrance plays a key role in the diastereoselectivity of the reaction.

Probing the role of an invariant active site His in family GH1 ß-glycosidases.

The reaction mechanism of glycoside hydrolases belonging to family 1 (GH1) of carbohydrate-active enzymes classification, hydrolysing ß-O-glycosidic bonds, is well characterised. This family includes several thousands of enzymes with more than 20 different EC numbers depending on the sugar glycone recognised as substrate. Most GH1 ß-glycosidases bind their substrates with similar specificity through invariant amino acid residues. Despite extensive studies, the clear identification of the roles played by each of these residues in the recognition of different glycones is not always possible. We demonstrated here that a histidine residue, completely conserved in the active site of the enzymes of this family, interacts with the C2-OH of the substrate in addition to the C3-OH as previously shown by 3 D-structure determination.