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.

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.

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.

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.

Computational modeling of carbohydrate processing enzymes reactions.

Carbohydrate processing enzymes are of biocatalytic interest. Glycoside hydrolases and the recently discovered lytic polysaccharide monooxygenase for their use in biomass degradation to obtain biofuels or valued chemical entities. Glycosyltransferases or engineered glycosidases and phosphorylases for the synthesis of carbohydrates and glycosylated products. Quantum mechanics-molecular mechanics (QM/MM) methods are highly contributing to establish their different chemical reaction mechanisms. Other computational methods are also used to study enzyme conformational changes, ligand pathways, and processivity, e.g. for processive glycosidases like cellobiohydrolases. There is still a long road to travel to fully understand the role of conformational dynamics in enzyme activity and also to disclose the variety of reaction mechanisms these enzymes employ. Additionally, computational tools for enzyme engineering are beginning to be applied to evaluate substrate specificity or aid in the design of new biocatalysts with increased thermostability or tailored activity, a growing field where molecular modeling is finding its way.

Entrapment of enzyme aggregates in chitosan beads for aroma release in white wines.

Glycosidases are enzymes involved in the cascade reactions leading to the release of aromatic compounds in white wines. However, the use of commercial soluble glycosidases is facing difficulties due to their fast inactivation, poor reaction control, low efficiency of enzyme use, and the presence of catalyst residues in the product. Co-immobilization as cross-linked enzyme aggregates (combi-CLEAs) is a sound alternative allowing the immobilization of enzymes in their own protein matrix, yielding highly stable and active biocatalysts. Notwithstanding, their micrometer sized particles limit their application in industrial processes. To overcome this, combi-CLEAs of ß-D-glucosidase (ßG) and α-L-arabinofuranosidase (ARA) were entrapped in polymeric chitosan beads. The effect of crosslinking reagents and crosslinking time on the specific activity and stability of combi-CLEAs was studied, and the best conditions for the entrapment of the combi-CLEAs in polymeric chitosan beads were determined varying the concentration of the chitosan solution and the pH of the gelation agent solution. The resulting biocatalyst beads (average diameter 1.24 mm), retained full activity after 91 days of incubation under winemaking conditions, having specific activities of 0.91 and 0.88 international units of activity per gram for ßG and ARA, respectively. Such characteristics make them suitable for aroma enhancement in wines.

Glioma infiltration and extracellular matrix: key players and modulators.

An outstanding characteristic of gliomas is their infiltration into brain parenchyma, a property that impairs complete surgical resection; consequently, these tumors might recur, resulting in a high mortality rate. Gliomas invade along preferential routes, such as those along white matter tracts and in the perineuronal and perivascular spaces. Brain extracellular components and their partners and modulators play a crucial role in glioma cell invasion. This review presents an extensive survey of the literature, showing how the brain extracellular matrix (ECM) is modulated during the glioma infiltration process. We explore aspects of ECM interaction with glioma cells, reviewing the main glycosaminoglycans, glycoproteins and proteoglycans. We discuss the roles of ECM-binding proteins, including CD44, RHAMM, integrins and axonal guidance molecules, and highlight the role of proteases and glycosidases in glioma infiltration; in binding and release chemokines, cytokines and growth factors; and in generating new bioactive ECM fragments. We also consider the roles of cytoskeletal signaling, angiogenesis, miRNAs and the glial-to-mesenchymal transition linked to glioma invasion. We closely discuss therapeutic approaches based on the modulation of the extracellular matrix, targeting the control of glioma infiltration, its relative failure in clinical trials, and potential means to overcome this difficulty.

Identity and role of the non-conserved acid/base catalytic residue in the GH29 fucosidase from the spider Nephilingis cruentata

a-L-Fucosidases are widely occurring enzymes that remove fucose residues from N- and O-fucosylated glycoproteins. Comparison of amino acid sequences of fucosidases reveals that although the nucleophile is conserved among all a-L-fucosidases, the position of the acid/base residue is quite variable. Although several site-directed mutation studies have previously been performed on bacterial fucosidases, the only eukaryotic fucosidase so studied was the human fucosidase. Recent alignments indicate that human and Arthropoda a-L-fucosidases share at least 50% identity and the acid/base residue seems to be conserved among them suggesting a common acid/base residue in Metazoa. Here we describe the cloning and expression in Pichia pastoris of a very active a-L-fucosidase from the spider Nephilingis cruentata (NcFuc) with a Km value for pNPFuc of 0.4 mM. NcFuc hydrolyzed fucoidan, 2'fucosyllactose and also lacto-N-difucohexaose II. Mutants modified at the conserved residues D214N, E209A, E59A were expressed and characterized. The 500-fold lower kcat of D214N than the wild type was consistent with a role in catalysis, as was the 8000-fold lower kcat value of E59A. This was supported by the 57-fold increase in the kcat of E59A upon addition of azide. A complex pH/rate profile was seen for the wild-type and mutant forms of NcFuc, similar to those measured previously for the Sulfolobus fucosidase. The non-conservative catalytic structure and distinct active site organization reinforce the necessity of structural studies of new fucosidases.

Epididymal α-l-fucosidase and its possible role in remodelling the surface of bull spermatozoa.

The mammalian epididymis provides an appropriate environment for sperm maturation. During the epididymal transit, spermatozoa undergo biochemical and morphological changes that lead to the acquisition of the fertilizing capacity. In this study we analysed the fucosylation status of membrane glycoproteins in the spermatozoa obtained from different regions of the bull epididymis. High amounts of fucose were detected on caput spermatozoa (R.F.I. = 1010 ± 20.35), mostly located in the post-acrosome zone. A significant decrease in the fucose levels was detected toward the cauda (R.F.I. = 540.5 ± 49.93) (P < 0.05). This decrease was in line with the increased activity of α-l-fucosidase in the cauda fluid. In sperm from the cauda, the defucosylation occurred in some proteins, whereas others showed higher fucosylation rates. A significant decrease of fucose in the gametes was observed upon incubation of crude cauda fluid with caput spermatozoa (from R.F.I. = 1.45 ± 0.08 to 1.06 ± 0.03) (P < 0.05) indicating that the α-l-fucosidase present in the epididymal fluid is active on spermatozoa. Moreover, this effect was blocked with specific enzyme inhibitors. These results provide direct evidence that the α-l-fucosidase from epididymal fluid participates in the fucose removal from spermatozoa, as a step of sperm maturation in the bull epididymis.

Nf-GH, a glycosidase secreted by Naegleria fowleri, causes mucin degradation: an in vitro and in vivo study.

AIM: The aim of this work was to identify, characterize and evaluate the pathogenic role of mucinolytic activity released by Naegleria fowleri. MATERIALS & METHODS: Zymograms, protease inhibitors, anion exchange chromatography, MALDI-TOF-MS, enzymatic assays, Western blot, and confocal microscopy were used to identify and characterize a secreted mucinase; inhibition assays using antibodies, dot-blots and mouse survival tests were used to evaluate the mucinase as a virulence factor. RESULTS: A 94-kDa protein with mucinolytic activity was inducible and abolished by p-hydroxymercuribenzoate. MALDI-TOF-MS identified a glycoside hydrolase. Specific antibodies against N. fowleri-glycoside hydrolase inhibit cellular damage and MUC5AC degradation, and delay mouse mortality. CONCLUSION: Our findings suggest that secretory products from N. fowleri play an important role in mucus degradation during the invasion process.

Differential distribution and biochemical characteristics of hydrolases among developmental stages of Schistosoma mansoni may offer new anti-parasite targets.

Schistosoma mansoni enzymes play important roles in host-parasite interactions and are potential targets for immunological and/or pharmacological attack. The aim of this study was to comparatively assess the presence of hydrolytic activities (phosphatases, glycosidases, aminopeptidases) in soluble (SF) and membrane (MF) fractions from different S. mansoni developmental stages (schistosomula 0 and 3h, juveniles, and adult worms of 28 and 45days-old, respectively), by using simple enzyme-substrate microassays. Our results show and confirm the prominent presence of alkaline phosphatase (AlP) activity in the MF of all the above parasite stages, highlighting also the relevant presence of MF-associated α-mannosidase (α-MAN) activity in juveniles. A soluble AlP activity, together with ß-N-D-acetylglucosaminidase (ß-NAG), and α-MAN activities, was detected in SF of schistosomulum 0h. Soluble ß-NAG, α-MAN, acid phosphatase (AcP), leucin (LAP) and alanine (AAP) aminopeptidase activities were also seen in the SF of the other different developmental stages. This work shows different soluble and membrane-associated hydrolytic capacities in each S. mansoni developmental stage from schistosomula to adults that might be exploitable as potential new targets for immune and/or chemoprophylactic strategies.

Association between three glycosidases activity A - mannosidase ( A-ΜΑΝ ), B-Ν-acetyloglucosaminidase (NAGASE) and B - galactosidase (B-GAL) andin vitro fertilization of bovine oocytes collected from different-sized follicles

We studied the role of three glycosidases (α-mannosidase - α-ΜΑΝ, β-N-acetyloglucosaminidase -NAGASE and β-galactosidase - β-GAL) in follicularfluid (FF) and in fertilization medium (FM) of bovineoocytes. Oocytes were allocated into 3 groups accordingto the follicular size (controls - CF: 2-8 mm, smallfollicle group - SF: 2-5 mm, large follicle group - LF:>5-8 mm). Bovine embryos were produced in vitroeither in groups (experiment 1, n = 2099 oocytes) orindividually (experiment 2, n = 79 oocytes). In bothexperiments, the activity of all glycosidases in the FF oflarge follicles was significantly lower than in the FF ofsmall follicles group. In the FM of LF-group oocytes, α-MAN and NAGASE were significantly higher comparedto SF- and CF-group oocytes (experiment 1) and β-GALwas significantly higher in SF- compared to CF-groupoocytes (experiment 2). Cleavage rate was similar amongall groups in both experiments; however significantlyhigher blastocyst formation was noted in CF-groupcompared to LF- (days 7, 8, 9) and SF- (days 8, 9) groups(experiment 1). In follicular fluid of small follicle group,β-GAL was associated positively with degeneratingoocytes’ number and negatively with blastocyst rate atdays 7, 8 (P = 0.065) and 9 (experiment 1). In fertilizationmedium of control group, α-MAN related negatively tocleavage rate (P < 0.05) and β-GAL to blastocyst rate atday 8 (P = 0.089) or day 9 (P = 0.072) (experiment 1).During fertilization, in experiment 1 all oocytesconsumed β-GAL and only control or small follicleoocytes consumed α-MAN; in experiment 1, only largefollicle oocytes released NAGASE, whereas all oocytesreleased all three glycosidases in experiment 2. Inconclusion, glycosidases affect the developmentalcompetence of oocytes collected from different sizedfollicles during in vitro fertilization, performed either ingroups or individually; their role in follicular fluid isdifferent from that in fertilization medium.

Association between three glycosidases activity A - mannosidase ( A-ΜΑΝ ), B-Ν-acetyloglucosaminidase (NAGASE) and B - galactosidase (B-GAL) andin vitro fertilization of bovine oocytes collected from different-sized follicles

We studied the role of three glycosidases (α-mannosidase - α-ΜΑΝ, β-N-acetyloglucosaminidase -NAGASE and β-galactosidase - β-GAL) in follicularfluid (FF) and in fertilization medium (FM) of bovineoocytes. Oocytes were allocated into 3 groups accordingto the follicular size (controls - CF: 2-8 mm, smallfollicle group - SF: 2-5 mm, large follicle group - LF:>5-8 mm). Bovine embryos were produced in vitroeither in groups (experiment 1, n = 2099 oocytes) orindividually (experiment 2, n = 79 oocytes). In bothexperiments, the activity of all glycosidases in the FF oflarge follicles was significantly lower than in the FF ofsmall follicles group. In the FM of LF-group oocytes, α-MAN and NAGASE were significantly higher comparedto SF- and CF-group oocytes (experiment 1) and β-GALwas significantly higher in SF- compared to CF-groupoocytes (experiment 2). Cleavage rate was similar amongall groups in both experiments; however significantlyhigher blastocyst formation was noted in CF-groupcompared to LF- (days 7, 8, 9) and SF- (days 8, 9) groups(experiment 1). In follicular fluid of small follicle group,β-GAL was associated positively with degeneratingoocytes number and negatively with blastocyst rate atdays 7, 8 (P = 0.065) and 9 (experiment 1). In fertilizationmedium of control group, α-MAN related negatively tocleavage rate (P < 0.05) and β-GAL to blastocyst rate atday 8 (P = 0.089) or day 9 (P = 0.072) (experiment 1).During fertilization, in experiment 1 all oocytesconsumed β-GAL and only control or small follicleoocytes consumed α-MAN; in experiment 1, only largefollicle oocytes released NAGASE, whereas all oocytesreleased all three glycosidases in experiment 2. Inconclusion, glycosidases affect the developmentalcompetence of oocytes collected from different sizedfollicles during in vitro fertilization, performed either ingroups or individually; their role in follicular fluid isdifferent from that in fertilization medium.(AU)

Aroma Release in Wine Using Co-Immobilized Enzyme Aggregates.

Aroma is a remarkable factor of quality and consumer preference in wine, representing a distinctive feature of the product. Most aromatic compounds in varietals are in the form of glycosidic precursors, which are constituted by a volatile aglycone moiety linked to a glucose residue by an O-glycosidic bond; glucose is often linked to another sugar (arabinose, rhamnose or apiose). The use of soluble ß-glycosidases for aroma liberation implies the addition of a precipitating agent to remove it from the product and precludes its reuse after one batch. An attractive option from a technological perspective that will aid in removing such constraints is the use of immobilized glycosidases. Immobilization by aggregation and crosslinking is a simple strategy producing enzyme catalysts of very high specific activity, being an attractive option to conventional immobilization to solid inert supports. The purpose of this work was the evaluation of co-immobilized ß-glycosidases crosslinked aggregates produced from the commercial preparation AR2000, which contains the enzymes involved in the release of aromatic terpenes in Muscat wine (α-l-arabinofuranosidase and ß-d-glucopyranosidase). To do so, experiments were conducted with co-immobilized crosslinked enzyme aggregates (combi-CLEAs), and with the soluble enzymes, using an experiment without enzyme addition as control. Stability of the enzymes at the conditions of winemaking was assessed and the volatiles composition of wine was determined by SPE-GC-MS. Stability of enzymes in combi-CLEAs was much higher than in soluble form, 80% of the initial activity remaining after 60 days in contact with the wine; at the same conditions, the soluble enzymes had lost 80% of their initial activities after 20 days. Such higher stabilities will allow prolonged use of the enzyme catalyst reducing its impact in the cost of winemaking. Wine treated with combi-CLEAs was the one exhibiting the highest concentration of total terpenes (18% higher than the control) and the highest concentrations of linalool (20% higher), nerol (20% higher) and geraniol (100% higher), which are the most important terpenes in determining Muscat typicity. Co-immobilized enzymes were highly stable at winemaking conditions, so their reutilization is possible and technologically attractive by reducing the impact of enzyme cost on winemaking cost.

Seminal plasma hexosaminidase in patients with normal and abnormal spermograms.

BACKGROUND: Glycosidases profusion in male reproductive fluids suggests a possible relationship with sperm function. Although Hexosaminidase (Hex) is the most active glycosidase in epididymal fluid and seminal plasma, as well as in spermatozoa, Glucosidase is considered a marker for epididymal function and azoospermia. OBJECTIVE: The aim of this study was to determine Hex activity in seminal plasma from patients with normal and abnormal spermograms and analyze its correlation with seminal parameters. MATERIALS AND METHODS: In this cross sectional study, seminal plasma from azoospermic, asthenozoospermic, teratozoospermic, and normozoospermic patients was analyzed for the activity of: total Hex, HexA isoform, and glucosidase. Besides, hexosamine levels were determined, and the amount of Hex was quantified by immunoblot with a specific antibody. Correlation of Hex activity with seminal parameters was also analyzed. RESULTS: Hex activity, like glucosidase, was significantly reduced in azoospermic samples (44, 49, and 60% reduction for total Hex, HexA and glucosidase, respectively). A reduced amount of Hex in azoospermic samples was confirmed by western immunoblot. Hex activity was negatively correlated with round cells in azoospermic samples and positively correlated with motility in asthenozoospermic ones. CONCLUSION: The results suggested that Hex activity was reduced in azoospermic samples and this was due to a lower amount of enzyme. The correlation to seminal parameters related to particular pathologies suggests a possible relationship of Hex with fertilizing capacity.

Functional characterization of Sporothrix schenckii glycosidases involved in the N-linked glycosylation pathway.

Protein glycosylation pathways are conserved metabolic processes in eukaryotic organisms and are required for cell fitness. In fungal pathogens, the N-linked glycosylation pathway is indispensable for proper cell wall composition and virulence. In Sporothrix schenckii sensu stricto, the causative agent of sporotrichosis, little is known about this glycosylation pathway. Here, using a genome-wide screening for putative members of the glycosyl hydrolase (CAZy - GH) families 47 and 63, which group enzymes involved in the processing step during N-linked glycan maturation, we found seven homologue genes belonging to family 47 and one to family 63. The eight genes were individually expressed in C. albicans null mutants lacking either MNS1 (for members of family 47) or CWH41 (for the member of family 63). Our results indicate that SsCWH41 is the functional ortholog of CaCWH41, whereas SsMNS1 is the functional ortholog of CaMNS1. The remaining genes of family 47 encode Golgi mannosidases and endoplasmic reticulum degradation-enhancing alpha-mannosidase-like proteins (EDEMs). Since these GH families gather proteins used as target for drugs to control cell growth, identification of these genes could help in the design of antifungals that could be used to treat sporotrichosis and other fungal diseases. In addition, to our knowledge, we are the first to report that Golgi mannosidases and EDEMs are expressed and characterized in yeast cells.

Aroma enhancement in wines using co-immobilized Aspergillus niger glycosidases.

A major fraction of monoterpenes and norisoprenoids in young wines is conjugated to sugars representing a significant reservoir of aromatic precursors. To promote their release, ß-glucosidase, α-arabinosidase, and α-rhamnosidase from a commercial Aspergillus niger preparation, were immobilized onto acrylic beads. The aim of this work was the development and application of an immobilized biocatalyst, due to the well-known advantages over soluble enzyme preparations: control of the reaction progress and preparation of enzyme-free products. In addition, the obtained derivative showed increased stability in simile wine conditions. After the treatment of Muscat wine with the biocatalyst for 20days, free monoterpenes increased significantly (from 1119 to 2132µg/L, p<0.01) with respect to the control wine. Geraniol was increased 3,4-fold over its flavor thresholds, and accordingly its impact on sensorial properties was very relevant: nine of ten judges considered treated wine more intense in fruit and floral notes.

Evidence of ternary complex formation in Trypanosoma cruzi trans-sialidase catalysis.

Trypanosoma cruzi trans-sialidase (TcTS) is a key target protein for Chagas disease chemotherapy. In this study, we investigated the implications of active site flexibility on the biochemical mechanism of TcTS. Molecular dynamics studies revealed remarkable plasticity in the TcTS catalytic site, demonstrating, for the first time, how donor substrate engagement with the enzyme induces an acceptor binding site in the catalytic pocket that was not previously captured in crystal structures. Furthermore, NMR data showed cooperative binding between donor and acceptor substrates, supporting theoretical results. In summary, our data put forward a coherent dynamic framework to understand how a glycosidase evolved its highly efficient trans-glycosidase activity.

Glicosidases e sulfatases no molusco marinho aplysia cervina / Glycosidases and sulfatases from the marine mollusc aplysia cervina

RESUMO Glicosaminoglicanos (GAGs) são carboidratos presentes em todos os organismos que apresentam organização tissular. Glicosidases e sulfatases são as enzimas envolvidas na degradação destes compostos em vertebrados e invertebrados. O objetivo deste trabalho foi identificar a presença destas enzimas em diferentes tecidos do molusco Aplysiacervina. O molusco foi dissecado em esôfago, fígado, moela e músculo e homogeneizados separadamente. Após centrifugação os sobrenadantes (extratos brutos) foram fracionados com sulfato de amônio nas concentrações de 0-30% (F-I), 30-50% (FII) e 50-80% (FIII). Foi identificada a presença de sulfatase, β-glucuronidase, β-D-N-acetilgalactosaminidase e α-glucosaminidase nos extratos brutos de todos os tecidos investigados. A enzima α-fucosidase não foi detectada em esôfago, nos extratos brutos e F-I de moela, mas sim nas frações de fígado e músculo. A sulfatase apresentou-se mais ativa na F-III de fígado e moela com atividades específicas de 3,0 e 4,8, respectivamente. A α-glucosaminidase foi mais ativa nas frações F-III de esôfago e músculo apresentando atividades específicas de 3,9 e 2,0, respectivamente. Estes resultados indicam a existência de atividade catabólica sobre os GAGs neste invertebrado marinho.

ABSTRACT Glycosaminoglycans (GAGs) are carbohydrates present in all organisms with tissue organization. Glycosidases and sulfatases are the enzymes involved in their degradation in vertebrates and invertebrates. The objective of this work was to identify the presence of these enzymes in different tissues of the mollusc Aplysia cervina. The mollusc was dissected, separating out the esophagus, liver, gizzard and muscle, which were homogenized separately. After centrifugation the supernatants (crude extracts) were fractionated with ammonium sulfate in the concentrations of 0-30% (F-I), 30-50% (FII) and 50-80% (FIII). The presence of the sulfatase,β-glucuronidase, β-D-N-acetilgalactosaminidase and α-glucosaminidase was identified in the crude extracts of all studied tissues. α-fucosidasewasnot found in the esophagus or in the crude extracts and F-I from gizzard, but was detected in the fractions from liver and muscle. Sulfatase was more active in F-III from liver and gizzard, demonstrating specific activity of the 3.0 and 4.8, respectively. α-glucosaminidase was more active in the F-III fraction from esophagus and muscle presenting 3.9 and 2.0 specifics activities, respectively. These results indicate the existence of catabolic activity on GAGs in these marine invertebrate.