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.

Valuation of the significant hypoglycemic activity of black currant anthocyanin extract by both starch structure transformation and glycosidase activity inhibition.

The objective of this study was to investigate the impact of anthocyanin-rich black currant extract (BCE) on the structural properties of starch and the inhibition of glycosidases, gathering data and research evidence to support the use of low glycemic index (GI) foods. The BCE induced a change in the starch crystal structure from A-type to V-type, resulting in a drop in digestibility from 81.41 % to 65.57 %. Furthermore, the inhibitory effects of BCE on glycosidases activity (α-glucosidase: IC50 = 0.13 ± 0.05 mg/mL and α-amylase: IC50 = 2.67 ± 0.16 mg/mL) by inducing a change in spatial conformation were confirmed through in vitro analysis. The presence of a 5'-OH group facilitated the interaction between anthocyanins and receptors of amylose, α-amylase, and α-glucosidase. The glycosyl moiety enhanced the affinity for amylose yet lowered the inhibitory effect on α-amylase. The in vivo analysis demonstrated that BCE resulted in a reduction of 3.96 mM·h in blood glucose levels (Area Under Curve). The significant hypoglycemic activity, particularly the decrease in postprandial blood glucose levels, highlights the potential of utilizing BCE in functional foods for preventing diabetes.

Kinetics and molecular modeling studies on the inhibition mechanism of GH13 α-glycosidases by small molecule ligands.

Alpha-glucosidase inhibitors play an important role in Diabetes Mellitus (DM) treatment since they prevent postprandial hyperglycemia. The Glycoside Hydrolase family 13 (GH13) is the major family of enzymes acting on substrates containing α-glucoside linkages, such as maltose and amylose/amylopectin chains in starch. Previously, our group identified glycoconjugate 1H-1,2,3-triazoles (GCTs) inhibiting two GH13 α-glycosidases: yeast maltase (MAL12) and porcine pancreatic amylase (PPA). Here, we combined kinetic studies and computational methods on nine GCTs to characterize their inhibitory mechanism. They all behaved as reversible inhibitors, and kinetic models encompassed noncompetitive and various mechanisms of mixed-type inhibition for both enzymes. Most potent inhibitors displayed Ki values of 30 µM for MAL12 (GPESB16) and 37 µM for PPA (GPESB15). Molecular dynamics and docking simulations indicated that on MAL12, GPESB15 and GPESB16 bind in a cavity adjacent to the active site, while on the PPA, GPESB15 was predicted to bind at the entrance of the catalytic site. Notably, despite its putative location within the active site, the binding of GPESB15 does not obstruct the substrate's access to the cleavage site. Our study contributes to paving the way for developing novel therapeutic strategies for managing DM-2 through GH13 α-glycosidases inhibition.

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.

Characterization of Solitalea canadensis α-mannosidase with specific activity towards α1,3-Mannosidic linkages.

A recombinant exo-α-mannosidase from Solitalea canadensis (Sc3Man) has been characterized to exhibit strict specificity for hydrolyzing α1,3-mannosidic linkages located at the non-reducing end of glycans containing α-mannose. Enzymatic characterization revealed that Sc3Man operates optimally at a pH of 5.0 and at a temperature of 37 °C. The enzymatic activity was notably enhanced twofold in the presence of Ca2+ ions, emphasizing its potential dependency on this metal ion, while Cu2+ and Zn2+ ions notably impaired enzyme function. Sc3Man was able to efficiently cleave the terminal α1,3 mannose residue from various high-mannose N-glycan structures and from the model glycoprotein RNase B. This work not only expands the categorical scope of bacterial α-mannosidases, but also offers new insight into the glycan metabolism of S. canadensis, highlighting the enzyme's utility for glycan analysis and potential biotechnological applications.

Structural insights into peptidoglycan glycosidase EtgA binding to the inner rod protein EscI of the type III secretion system via a designed EscI-EtgA fusion protein.

Bacteria express lytic enzymes such as glycosidases, which have potentially self-destructive peptidoglycan (PG)-degrading activity and, therefore, require careful regulation in bacteria. The PG glycosidase EtgA is regulated by localization to the assembling type III secretion system (T3SS), generating a hole in the PG layer for the T3SS to reach the outer membrane. The EtgA localization was found to be mediated via EtgA interacting with the T3SS inner rod protein EscI. To gain structural insights into the EtgA recognition of EscI, we determined the 2.01 Å resolution structure of an EscI (51-87)-linker-EtgA fusion protein designed based on AlphaFold2 predictions. The structure revealed EscI residues 72-87 forming an α-helix interacting with the backside of EtgA, distant from the active site. EscI residues 56-71 also were found to interact with EtgA, with these residues stretching across the EtgA surface. The ability of the EscI to interact with EtgA was also probed using an EscI peptide. The EscI peptide comprising residues 66-87, slightly larger than the observed EscI α-helix, was shown to bind to EtgA using microscale thermophoresis and thermal shift differential scanning fluorimetry. The EscI peptide also had a two-fold activity-enhancing effect on EtgA, whereas the EscI-EtgA fusion protein enhanced activity over four-fold compared to EtgA. Our studies suggest that EtgA regulation by EscI could be trifold involving protein localization, protein activation, and protein stabilization components. Analysis of the sequence conservation of the EscI EtgA interface residues suggested a possible conservation of such regulation for related proteins from different bacteria.

Structural Characteristics and Antioxidant Activity Analysis of Polysaccharides from Pinelliae Rhizoma and Its Processed Products Before and After Hydrolysis （Enzymolysis） by Sugar Spectrum / 中国实验方剂学杂志

ObjectiveThe glycosidic linkage structural characteristics of polysaccharides from Pinelliae Rhizoma（PR） and its processed products were analyzed by sugar spectrum， high performance thin layer chromatography（HPTLC）， fluorescence-assisted carbohydrate gel electrophoresis（PACE） based on partial acid hydrolysis and specific glycosidase hydrolysis， and the antioxidant activities of polysaccharides before and after hydrolysis（enzymolysis） were compared. MethodPolysaccharides from PR and its processed products were extracted by ultrasound extraction， starch was hydrolyzed by α-amylase， and small molecules below 3 kDa were removed by ultrafiltration. The purified polysaccharides were prepared by hydrolysis of acid and five different specific glycosidases， and the hydrolysates were analyzed by HPTLC and PACE. The antioxidant capacity of polysaccharides was analyzed by 2，2′-azino-bis（3-ethylbenzthiazoline-6-sulfonic acid）（ABTS） and 2，2-diphenyl-1-picrylhydrazyl（DPPH） free radical scavenging experiment before and after different hydrolysis. ResultThrough HPTLC and PACE analysis， it was found that polysaccharides from PR and its processed products could be hydrolyzed by β-galactosidase， β-mannase， cellulase and pectinase， but hardly hydrolyzed by glucanase， indicating that the polysaccharides contained β-galactopyranoside bond， β-1，4-mannoside bond， β-1，4-glucoside bond and α-1，4-galacturonic acid glycosidic bond. In vitro antioxidant experiments showed that the ABTS radical scavenging capacity of the polysaccharides from PR and its processed products was weakened after acid hydrolysis and pectinase enzymatic hydrolysis， while the ABTS radical scavenging capacity was enhanced after enzymatic hydrolysis with cellulase， β-galactosidase， and β-mannase. And after different hydrolysis， the DPPH free radical scavenging capacity of polysaccharides from PR and its processed products was all significantly enhanced. ConclusionThe glycosidic linkage structural characteristics of polysaccharides from PR and its processed products was analyzed by sugar spectrum in this paper， and the relationship between glycosidic bond types and their antioxidant activity was clarified through in vitro antioxidant experiments， which is beneficial for further elucidating the material basis of the related efficacy of PR and its processed products， and providing new ideas and methods for analyzing the structural characteristics of polysaccharides in Chinese medicines.

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.

Fungal Glycosidases in Sporothrix Species and Candida albicans.

Glycoside hydrolases (GHs) are enzymes that participate in many biological processes of fungi and other organisms by hydrolyzing glycosidic linkages in glycosides. They play fundamental roles in the degradation of carbohydrates and the assembly of glycoproteins and are important subjects of studies in molecular biology and biochemistry. Based on amino acid sequence similarities and 3-dimensional structures in the carbohydrate-active enzyme (CAZy), they have been classified in 171 families. Members of some of these families also exhibit the activity of trans-glycosydase or glycosyl transferase (GT), i.e., they create a new glycosidic bond in a substrate instead of breaking it. Fungal glycosidases are important for virulence by aiding tissue adhesion and colonization, nutrition, immune evasion, biofilm formation, toxin release, and antibiotic resistance. Here, we review fungal glycosidases with a particular emphasis on Sporothrix species and C. albicans, two well-recognized human pathogens. Covered issues include a brief account of Sporothrix, sporotrichosis, the different types of glycosidases, their substrates, and mechanism of action, recent advances in their identification and characterization, their potential biotechnological applications, and the limitations and challenges of their study given the rather poor available information.

The Role of a Loop in the Non-catalytic Domain B on the Hydrolysis/Transglycosylation Specificity of the 4-α-Glucanotransferase from Thermotoga maritima.

The mechanism by which glycoside hydrolases control the reaction specificity through hydrolysis or transglycosylation is a key element embedded in their chemical structures. The determinants of reaction specificity seem to be complex. We looked for structural differences in domain B between the 4-α-glucanotransferase from Thermotoga maritima (TmGTase) and the α-amylase from Thermotoga petrophila (TpAmylase) and found a longer loop in the former that extends towards the active site carrying a W residue at its tip. Based on these differences we constructed the variants W131G and the partial deletion of the loop at residues 120-124/128-131, which showed a 11.6 and 11.4-fold increased hydrolysis/transglycosylation (H/T) ratio relative to WT protein, respectively. These variants had a reduction in the maximum velocity of the transglycosylation reaction, while their affinity for maltose as the acceptor was not substantially affected. Molecular dynamics simulations allow us to rationalize the increase in H/T ratio in terms of the flexibility near the active site and the conformations of the catalytic acid residues and their associated pKas.

Momordica charantia seed proteins - Purification, biochemical characterization of a class II α-mannosidase isoenzyme and its interaction with the lectin and protein body membrane.

Momordica charantia seeds contain a galactose specific lectin and mixture of glycosidases. These bind to lectin-affigel at pH 5.0 and are all eluted at pH 8.0. From the mixture, α-mannosidase was separated by gel filtration (purified enzyme Mr âˆ¼ 238 kDa). In native PAGE (silver staining) it showed three bands that stained with methylumbelliferyl substrate (possible isoforms). Ion exchange chromatography separated two isoforms in 0.5 M eluates and one isoform in 1.0 M eluate. In SDS-PAGE it dissociated to Mr ∼70 and 45 kDa subunits, showing antigenic similarity to jack bean enzyme. MALDI analysis confirmed the 70 kDa band to be α-mannosidase with sequence identity to the genomic sequence of Momordica charantia enzyme (score 83, 29 % sequence coverage). The pH, temperature optima were 5.0 and 60o C respectively. Kinetic parameters KM and Vmax estimated with p-nitrophenyl α-mannopyranoside were 0.85 mM and 12.1 U/mg respectively. Swainsonine inhibits the enzyme activity (IC50 value was 50 nM). Secondary structural analysis at far UV (190-300 nm) showed 11.6 % α-helix and 36.5 % ß-sheets. 2.197 mg of the enzyme was found to interact with 3.75 mg of protein body membrane at pH 5.0 and not at pH 8.0 suggesting a pH dependent interaction.

Evaluation of nonnatural L-iminosugar C,C-glycosides, a new class of C-branched iminosugars, as glycosidase inhibitors.

Capitalizing on a previously developed Staudinger/azaWittig/Grignard (SAWG)-ring contraction sequence that furnished protected six-membered L-iminosugar C,C-glycosides bearing an allyl group and various substituents at the pseudoanomeric position, the synthesis and glycosidase inhibition of a small library of six- and seven-membered L-iminosugar C,C-glycosides is reported. Their hydrogenolysis or cyclization by RCM followed by deprotection afforded eleven L-iminosugars including spirocyclic derivatives. All compounds adopt a 1C4 conformation in solution according to NMR data. Compared to previously reported branched L-iminosugars, the L-iminosugar C,C-glycosides reported herein were less potent glycosidase inhibitors. However, some of these compounds showed micromolar inhibition of human lysosome ß-glucocerebrosidase suggesting that such iminosugars could be useful to access potent CGase inhibitors by adjusting the structure/length of the pseudoanomeric substituents.

Glycoside hydrolases from (hyper)thermophilic archaea: structure, function, and applications.

(Hyper)thermophilic archaeal glycosidases are enzymes that catalyze the hydrolysis of glycosidic bonds to break down complex sugars and polysaccharides at high temperatures. These enzymes have an unique structure that allows them to remain stable and functional in extreme environments such as hot springs and hydrothermal vents. This review provides an overview of the current knowledge and milestones on the structures and functions of (hyper)thermophilic archaeal glycosidases and their potential applications in various fields. In particular, this review focuses on the structural characteristics of these enzymes and how these features relate to their catalytic activity by discussing different types of (hyper)thermophilic archaeal glycosidases, including ß-glucosidases, chitinase, cellulases and α-amylases, describing their molecular structures, active sites, and mechanisms of action, including their role in the hydrolysis of carbohydrates. By providing a comprehensive overview of (hyper)thermophilic archaeal glycosidases, this review aims to stimulate further research into these fascinating enzymes.

Gene expression dynamics of natural assemblages of heterotrophic flagellates during bacterivory.

BACKGROUND: Marine heterotrophic flagellates (HF) are dominant bacterivores in the ocean, where they represent the trophic link between bacteria and higher trophic levels and participate in the recycling of inorganic nutrients for regenerated primary production. Studying their activity and function in the ecosystem is challenging since most of the HFs in the ocean are still uncultured. In the present work, we investigated gene expression of natural HF communities during bacterivory in four unamended seawater incubations. RESULTS: The most abundant species growing in our incubations belonged to the taxonomic groups MAST-4, MAST-7, Chrysophyceae, and Telonemia. Gene expression dynamics were similar between incubations and could be divided into three states based on microbial counts, each state displaying distinct expression patterns. The analysis of samples where HF growth was highest revealed some highly expressed genes that could be related to bacterivory. Using available genomic and transcriptomic references, we identified 25 species growing in our incubations and used those to compare the expression levels of these specific genes. Video Abstract CONCLUSIONS: Our results indicate that several peptidases, together with some glycoside hydrolases and glycosyltransferases, are more expressed in phagotrophic than in phototrophic species, and thus could be used to infer the process of bacterivory in natural assemblages.

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.

Glycosidase mechanisms: Sugar conformations and reactivity in endo- and exo-acting enzymes.

The enzymatic breakdown of carbohydrates plays a critical role in several biological events and enables the development of sustainable processes to obtain bioproducts and biofuels. In this scenario, the design of efficient inhibitors for glycosidases that can act as drug targets and the engineering of carbohydrate-active enzymes with tailored catalytic properties is of remarkable importance. To guide rational approaches, it is necessary to elucidate enzyme molecular mechanisms, in particular understanding how the microenvironment modulates the conformational space explored by the substrate. Computer simulations, especially those based on ab initio methods, have provided a suitable atomic description of carbohydrate conformations and catalytic reactions in several glycosidase families. In this review, we will focus on how the active-site topology (pocket or cleft) and mode of cleavage (endo or exo) can affect the catalytic mechanisms adopted by glycosidases, in particular the substrate conformations along the reaction coordinate.

Biotransformation of Platycosides, Saponins from Balloon Flower Root, into Bioactive Deglycosylated Platycosides.

Platycosides, saponins from balloon flower root (Platycodi radix), have diverse health benefits, such as antioxidant, anti-inflammatory, anti-tussive, anti-cancer, anti-obesity, anti-diabetes, and whitening activities. Deglycosylated platycosides, which show greater biological effects than glycosylated platycosides, are produced by the hydrolysis of glycoside moieties in glycosylated platycosides. In this review, platycosides are classified according to the chemical structures of the aglycone sapogenins and also divided into natural platycosides, including major, minor, and rare platycosides, depending on the content in Platycodi radix extract and biotransformed platycosides. The biological activities of platycosides are summarized and methods for deglycosylation of saponins, including physical, chemical, and biological methods, are introduced. The biotransformation of glycosylated platycosides into deglycosylated platycosides was described based on the hydrolytic pathways of glycosides, substrate specificity of glycosidases, and specific productivities of deglycosylated platycosides. Methods for producing diverse and/or new deglycosylated platycosides are also proposed.

Role of Streptococcus pneumoniae extracellular glycosidases in immune evasion.

Streptococcus pneumoniae (pneumococcus) typically colonizes the human upper airway asymptomatically but upon reaching other sites of the host body can cause an array of diseases such as pneumonia, bacteremia, otitis media, and meningitis. Be it colonization or progression to disease state, pneumococcus faces multiple challenges posed by host immunity ranging from complement mediated killing to inflammation driven recruitment of bactericidal cells for the containment of the pathogen. Pneumococcus has evolved several mechanisms to evade the host inflicted immune attack. The major pneumococcal virulence factor, the polysaccharide capsule helps protect the bacteria from complement mediated opsonophagocytic killing. Another important group of pneumococcal proteins which help bacteria to establish and thrive in the host environment is surface associated glycosidases. These enzymes can hydrolyze host glycans on glycoproteins, glycolipids, and glycosaminoglycans and consequently help bacteria acquire carbohydrates for growth. Many of these glycosidases directly or indirectly facilitate bacterial adherence and are known to modulate the function of host defense/immune proteins likely by removing glycans and thereby affecting their stability and/or function. Furthermore, these enzymes are known to contribute the formation of biofilms, the bacterial communities inherently resilient to antimicrobials and host immune attack. In this review, we summarize the role of these enzymes in host immune evasion.

In silico screening-based discovery of inhibitors against glycosylation proteins dysregulated in cancer.

Targeting enzymes associated with the biosynthesis of aberrant glycans is an under-utilized strategy in discovering potential inhibitors or drugs against cancer. The formation of cancer-associated glycans is mainly due to the dysregulated expression of glycosyltransferases and glycosidases, which play crucial roles in maintaining cellular structure and function. We screened a database of more than 14,000 compounds consisting of natural products and drugs for inhibition against four glycosylation enzymes - Alpha1-6FucT, ST6Gal1, ERMan1, and GlcNAcT-V. The top inhibitors identified against each enzyme were subsequently analyzed for potential binding against all four enzymes. In silico screening results show several promising candidates that could potentially inhibit all four enzymes: (1) Amb20622156 (demethylwedelolactone) [ERMan1: -9.3 kcal/mol; Alpha1-6FucT: -7.3 kcal/mol; ST6Gal1: -8.4 kcal/mol; GlcNAcT-V: -7.2 kcal/mol], (2) Amb22173588 (1,2-dihydrotanshinone I) [ERMan1: -9.3 kcal/mol; Alpha1-6FucT: -6.1 kcal/mol; ST6Gal1: -9.2 kcal/mol; GlcNAcT-V: -7.9 kcal/mol], and (3) Amb22173591 (tanshinol B) [ERMan1: -9.3 kcal/mol; Alpha1-6FucT: -6.0 kcal/mol; ST6Gal1: -9.8 kcal/mol; GlcNAcT-V: -7.7 kcal/mol]. Drug-enzyme active site residue interaction analyses show that the putative inhibitors form non-covalent bonding interactions with key active site residues in each enzyme, suggesting critical target residues in the four enzymes' active sites. Furthermore, pharmacokinetic property prediction analysis using pkCSM indicates that all of these inhibitors have good ADMETox properties (i.e., log P < 5, Caco-2 permeability > 0.90, intestinal absorption > 30%, skin permeability>-2.5, CNS permeability <-3, maximum tolerated dose < 0.477, minnow toxicity<-0.3). The in silico docking approach to glycosylation enzyme inhibitor prediction could help guide and streamline the discovery of novel inhibitors against enzymes involved in aberrant protein glycosylation.Communicated by Ramaswamy H. Sarma.