Hydrolysis Of Glycosidic Bonds Research Articles

Construct a Catalyst using the Beta-Peptide Bundle

“What I cannot create, I do not understand.” Richard Feynman, 1988. Through the construction of an efficient catalyst using a non-natural material, this research project aims to deepen our understanding of the structural and energetic features that control catalysis. We expect our findings to inform the principles for the design and engineering of both proteinaceous enzymes and artificial catalysts. We set out to use beta-peptides - a unique yet virtually unexplored middle ground between proteins and small molecules - to devise catalysts for the formation or hydrolysis of glycosidic bonds. Beta-peptides are smaller and thus synthetically more tractable than natural proteins; nevertheless, they can still adopt higher order structures inaccessible to small molecules. For example, the Schepartz Laboratory reported previously that the EYYK β-peptide self-assembles into an octameric bundle that displays conformational flexibility and thermodynamic properties similar to natural protein. We therefore build our first generation design around the EYYK scaffold. Each beta-peptide bundle will be characterize by three biophysical techniques. In addition to structural information obtained by X-ray crystallography, two novel assays that evaluate the beta-peptide bundles based on their substrate binding affinity and reaction turnover rate will be carried out. Together these information provide details of the physical and chemical properties of the beta-peptides to guide the iterative design - synthesis - characterization process. The latest results of our first generation beta-peptide bundle, EYBK, will be presented. The utility of two newly developed assays for monitoring binding and turnover will also be discussed in the context of informing catalyst design.

Construction of a rice glycoside hydrolase phylogenomic database and identification of targets for biofuel research

Glycoside hydrolases (GH) catalyze the hydrolysis of glycosidic bonds in cell wall polymers and can have major effects on cell wall architecture. Taking advantage of the massive datasets available in public databases, we have constructed a rice phylogenomic database of GHs (http://ricephylogenomics.ucdavis.edu/cellwalls/gh/). This database integrates multiple data types including the structural features, orthologous relationships, mutant availability, and gene expression patterns for each GH family in a phylogenomic context. The rice genome encodes 437 GH genes classified into 34 families. Based on pairwise comparison with eight dicot and four monocot genomes, we identified 138 GH genes that are highly diverged between monocots and dicots, 57 of which have diverged further in rice as compared with four monocot genomes scanned in this study. Chromosomal localization and expression analysis suggest a role for both whole-genome and localized gene duplications in expansion and diversification of GH families in rice. We examined the meta-profiles of expression patterns of GH genes in twenty different anatomical tissues of rice. Transcripts of 51 genes exhibit tissue or developmental stage-preferential expression, whereas, seventeen other genes preferentially accumulate in actively growing tissues. When queried in RiceNet, a probabilistic functional gene network that facilitates functional gene predictions, nine out of seventeen genes form a regulatory network with the well-characterized genes involved in biosynthesis of cell wall polymers including cellulose synthase and cellulose synthase-like genes of rice. Two-thirds of the GH genes in rice are up regulated in response to biotic and abiotic stress treatments indicating a role in stress adaptation. Our analyses identify potential GH targets for cell wall modification.

Long Duration, Single-Molecule Monitoring of Lysozyme Kinetics

Tethering a single molecule of T4 lysoyzme to a carbon nanotube field effect transistor resulted in a novel technique for monitoring single-molecule enzymatic activity. Continuous electronic monitoring readily resolved lysozyme as a processive enzyme, since substrate release was clearly distinguished from serial hydrolysis of glycosidic bonds. On average, we observed hydrolysis of 100 consecutive bonds at 15 Hz rates when lysozyme processed natural peptidoglycan. Furthermore, long duration measurements allowed statistically-meaningful analysis of thousands of chemical events by the same molecule, uncovering seven independent timescales governing lysozyme's activity, including minute-by-minute dynamic disorder. Stability of the lysozyme-transistor method allowed these timescales to be studied as a function of environmental conditions such as substrate, pH, or temperature, in order to produce a detailed map of factors that affect single molecule processivity. For example, variations in pH do not change any of the rate constants of lysozyme's motions, but rather decrease enzyme activity by increasing the proportion of time stuck in an inactive, closed conformation. As another example, we found that peptidoglycan cross-links are directly responsible for a 50% drop in effective activity. Using a synthetic, linear substrate without cross-links, we observed long, uninterrupted processing runs to the end of individual substrate molecules. By comparison, lysozyme spends approximately half its time traversing the cross-links present in wild-type substrate. Rather than releasing substrate at a cross-link, however, lysozyme instead changes to an alternate, non-processive motion with which it can sidestep the cross-link. The combination of processive and non-processive motions allow lysozyme to remain effective and zigzag through wild-type substrate.

Metabolism of Saikosaponin a in Rats: Diverse Oxidations on the Aglycone Moiety in Liver and Intestine in Addition to Hydrolysis of Glycosidic Bonds

The main objective of the present study was to completely characterize the metabolites of the triterpenoid saikosaponin a (SSa) in rats. To this aim, we compared the metabolites in plasma, bile, urine, and feces samples following oral and i.v. routes of administration using liquid chromatography-diode array detector coupled with hybrid ion trap-time-of-flight mass spectrometry. As a result, besides 2 known metabolites, prosaikogenin f and saikogenin f, 15 new metabolites were detected in all. It was found that SSa is metabolized mainly in phase I manner, i.e., hydration and monooxidation on the aglycone moiety and hydrolysis of the β-glucosidic bond in the liver, and sequential hydrolysis of β-glucosidic and β-fucosidic bonds followed by dehydrogenation, hydroxylation, carboxylation, and combinations of these steps on the aglycone moiety in the intestinal tract. Both the renal and biliary routes were observed for the excretion of SSa and its metabolites. Further, a clear metabolic profile in rats was proposed in detail according to the results from the in vivo animal experiment after different routes of administration. Our results update the preclinical metabolism and disposition data on SSa, which is not only helpful for the future human metabolic study of this compound but also provides basic information for better understanding of the efficacy and safety of prescriptions containing saikosaponins.

Influenza Virus Neuraminidases with Reduced Enzymatic Activity That Avidly Bind Sialic Acid Receptors

Influenza virus neuraminidase (NA) cleaves off sialic acid from cellular receptors of hemagglutinin (HA) to enable progeny escape from infected cells. However, NA variants (D151G) of recent human H3N2 viruses have also been reported to bind receptors on red blood cells, but the nature of these receptors and the effect of the mutation on NA activity were not established. Here, we compare the functional and structural properties of a human H3N2 NA from A/Tanzania/205/2010 and its D151G mutant, which supports HA-independent receptor binding. While the wild-type NA efficiently cleaves sialic acid from both α2-6- and α2-3-linked glycans, the mutant exhibits much reduced enzymatic activity toward both types of sialosides. Conversely, while wild-type NA shows no detectable binding to sialosides, the D151G NA exhibits avid binding with broad specificity toward α2-3 sialosides. D151G NA binds the 3' sialyllactosamine (3'-SLN) and 6'-SLN sialosides with equilibrium dissociation constant (K(D)) values of 30.0 μM and 645 μM, respectively, which correspond to much higher affinities than the corresponding affinities (low mM) of HA to these glycans. Crystal structures of wild-type and mutant NAs reveal the structural basis for glycan binding in the active site by exclusively impairing the glycosidic bond hydrolysis step. The general significance of D151 among influenza virus NAs was further explored by introducing the D151G mutation into three N1 NAs and one N2 NA, which all exhibited reduced enzymatic activity and preferential binding to α2-3 sialosides. Since the enzymatic and binding activities of NAs are not routinely assessed, the potential for NA receptor binding to contribute to influenza virus biology may be underappreciated.

Model for Degradation of Galactoglucomannan in Hot Water Extraction Conditions

A kinetic analysis is presented for the homogeneous degradation of galactoglucomannan (GGM) in hot water extraction conditions. In the experiments used for constructing the kinetic model, GGM extracted from spruce wood meal was treated at temperatures from 150 to 170 °C and in buffered pH 3.8–4.2. The hydrolysis of glycosidic bonds in GGM was modeled as a random process which is catalyzed by hydrogen ions. The real hydrogen ion concentration at the reaction temperatures was estimated. The Arrhenius equation was used to describe the temperature dependence of the hydrolysis rate and an activation energy of 102 kJ/mol was obtained for the reaction. The presented model predicts accurately the evolution of the molecular weight distribution with lower computational load than other similar models.

Products from microwave and ultrasonic wave assisted acid hydrolysis of chitin

Hydrolysis of α-chitin in concentrated hydrochloric acid under elevated temperature is a general preparation of a nutrapharmaceutical glucosamine hydrochloride (GlcN.HCl). In this study, the microwave and ultrasonic wave assisted acid hydrolysis of shrimp shell α-chitin are investigated with an aim to improve the reaction rate and selectivity. Microwave heating shortens the hydrolysis time from 120min in the conventional heating process to 12min to afford GlcN.HCl in 57% yield. Sonication can be used to assist chitin dissolution in 38% HCl prior to the hydrolysis at 120°C for 120min to produce GlcN.HCl in 62% yield. The selective hydrolysis of glycosidic bond of chitin is achievable at 30°C for 4h to give N-acetyl glucosamine (GlcNAc) in 37% yield.

Single-molecule dynamics of lysozyme processing distinguishes linear and cross-linked peptidoglycan substrates.

The dynamic processivity of individual T4 lysozyme molecules was monitored in the presence of either linear or cross-linked peptidoglycan substrates. Single-molecule monitoring was accomplished using a novel electronic technique in which lysozyme molecules were tethered to single-walled carbon nanotube field-effect transistors through pyrene linker molecules. The substrate-driven hinge-bending motions of lysozyme induced dynamic electronic signals in the underlying transistor, allowing long-term monitoring of the same molecule without the limitations of optical quenching or bleaching. For both substrates, lysozyme exhibited processive low turnover rates of 20-50 s(-1) and rapid (200-400 s(-1)) nonproductive motions. The latter nonproductive binding events occupied 43% of the enzyme's time in the presence of the cross-linked peptidoglycan but only 7% with the linear substrate. Furthermore, lysozyme catalyzed the hydrolysis of glycosidic bonds to the end of the linear substrate but appeared to sidestep the peptide cross-links to zigzag through the wild-type substrate.

Mechanism of the alkali degradation of (6–4) photoproduct-containing DNA

The (6-4) photoproduct is one of the major damaged bases produced by ultraviolet light in DNA. This lesion is known to be alkali-labile, and strand breaks occur at its sites when UV-irradiated DNA is treated with hot alkali. We have analyzed the product obtained by the alkali treatment of a dinucleoside monophosphate containing the (6-4) photoproduct, by HPLC, NMR spectroscopy, and mass spectrometry. We previously found that the N3-C4 bond of the 5' component was hydrolyzed by a mild alkali treatment, and the present study revealed that the following reaction was the hydrolysis of the glycosidic bond at the 3' component. The sugar moiety of this component was lost, even when a 3'-flanking nucleotide was not present. Glycosidic bond hydrolysis was also observed for a dimer and a trimer containing 5-methyl-2-pyrimidinone, which was used as an analog of the 3' component of the (6-4) photoproduct, and its mechanism was elucidated. Finally, the alkali treatment of a tetramer, d(GT(6-4)TC), yielded 2'-deoxycytidine 5'-monophosphate, while 2'-deoxyguanosine 3'-monophosphate was not detected. This result demonstrated the hydrolysis of the glycosidic bond at the 3' component of the (6-4) photoproduct and the subsequent strand break by β-elimination. It was also shown that the glycosidic bond at the 3' component of the Dewar valence isomer was more alkali-labile than that of the (6-4) photoproduct.

Grafted Poly(1→4-β-glucan) Strands on Silica: A Comparative Study of Surface Reactivity as a Function of Grafting Density

Grafted poly(β-glucan) (β-glu) strands on the surface of silica are synthesized with varying degrees of grafting density, and display an amorphous-like environment via (13)C CP/MAS NMR spectroscopy. Thermal gravimetric analysis of these materials under oxidative conditions shows increased β-glu thermal stability with higher degrees of grafting density. The range of temperature stability between the most and least hydrogen-bound grafted β-glu strands spans 321 to 260 °C. This range is bound by the combustion temperature previously measured for crystalline and amorphous cellulose, with the former having greater oxidative stability, and is likely controlled by the extent of hydrogen bonding of a grafted β-glu strand with the underlying silica surface. When using these materials as reactants for glycosidic bond hydrolysis, the total number of reducing ends formed during reaction is quantified using a BCA colorimetric assay. Results demonstrate that the material with greatest interaction with silica surface silanols undergoes hydrolysis at an initial rate that is 6-fold higher than the material with the lowest degree of such interaction. The role of the surface as a reactive interface that can endow oxidative stability and promote hydrolysis activity has broad implications for surface-catalyzed processes dealing with biomass-derived polymers.

Glycoside Hydrolysis with Sugar-Templated Microgel Catalysts

Water-soluble, carbohydrate-templated microgels are explored as catalysts for the hydrolysis of glycosidic bonds. The reaction is more than 2 orders of magnitude accelerated for templated macromolecular catalysts over low-molecular-weight complexes. The structure of the carbohydrate used as template during material preparation directs the catalytic performance of the catalysts to differentiate epimeric substrates.

Direct Experimental Evidence for the High Chemical Reactivity of α‐ and β‐Xylopyranosides Adopting a 2,5B Conformation in Glycosyl Transfer

The effect of a (2,5)B boat conformation on xyloside reactivity has been investigated by studying the hydrolysis and glycosylation of a series of synthetic xyloside analogues based on a 2-oxabicyclo[2.2.2]octane framework, which forces the xylose analogue to adopt a (2,5)B conformation. The locked β-xylosides were found to hydrolyze 100-1200 times faster than methyl β-D-xylopyranoside, whereas the locked α-xylosides hydrolyzed up to 2×10(4) times faster than methyl α-D-xylopyranoside. A significant rate enhancement was also observed for the glycosylation reaction. The high reactivity of these conformers can be related to the imposition of a (2,5)B conformation, which approximates a transition state (TS) boat conformation. In this way, the energy penalty required to go from the chair to the TS conformation is already paid. These results parallel and support the observation that the GH-11 xylanase family force their substrate to adopt a (2,5)B conformation to achieve highly efficient enzymatic glycosidic bond hydrolysis.

Glycosidic-Bond Hydrolysis Mechanism Catalyzed by Cellulase Cel7A from Trichoderma reesei: A Comprehensive Theoretical Study by Performing MD, QM, and QM/MM Calculations

Cellulase Cel7A from Trichoderma reesei is one of the most abundant and effective cellulases. Structural studies have established that Cel7A is a retaining glycosidase and it can processively hydrolyze cellobiose units from the reducing end of a cellulose chain. Here, to elucidate the mechanism of enzymatic catalysis of cellulase Cel7A, we carried out a multisized level theoretical study by performing MD, QM, and QM/MM calculations. At the accurate level of theory, we showed the mechanism details of the catalytic cycle, which involves the configuration inversion of the anomeric center twice: the first results in the glycosidic bond cleavage and the formation of covalent glycosyl-enzyme intermediate, and the second restores the anomeric carbon to its original configuration. Calculated results have provided detailed structural and energetic information about these two processes, both of which proceed according to a S(N)2-type-like mechanism via loose transition state structures. It is clearly indicated that the glycosidic bond hydrolysis involves the formation of a covalent glycosyl-enzyme intermediate, which has been identified as the minimum on the potential energy surface. At the catalytic active region, hydrogen bond interactions exist throughout the whole process of the catalytic cycle, which are of special importance for stabilizing the glycosyl-enzyme intermediate. The present results provide a clear paradigm of the mechanisms of general glycosidases, which hydrolyze the glycosidic bonds with net retention of the anomeric configuration.

Unexpected phosphate salt-catalyzed hydrolysis of glycosidic bonds in model disaccharides: Cellobiose and maltose

Monobasic sodium phosphate salt is shown to unexpectedly catalyze cellobiose and maltose hydrolysis to glucose at temperatures between 90 °C and 120 °C and pH 4 in aqueous solution. A selectivity of up to 80% glucose is achieved, which increases with increasing disaccharide dilution. The catalytic role of the phosphate salt is evident in a comparison of the measured activation energy of the catalyzed process for cellobiose hydrolysis of 59 kJ mol −1 versus that reported for the background reaction of 136 kJ mol −1. The normalized rate of catalysis over background significantly increases further at higher pH. Zero-order dependence of hydrolysis rate on disaccharide concentration as well as a logarithmic dependence of hydrolysis rate on phosphate salt concentration is observed. A heterogeneous catalysis mechanism is proposed, which is supported by the presence of condensed phosphate in reaction solution via 31P NMR spectroscopy. This mechanism involves disaccharide chemisorption and subsequent activation via interactions with surface acid/base sites.

ChemInform Abstract: Antibody Catalysis of Glycosidic Bond Hydrolysis.

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Redox Mechanism of Glycosidic Bond Hydrolysis Catalyzed by 6-Phospho-α-glucosidase: A DFT Study

Glycosidic bonds are remarkably resistant to cleavage by chemical hydrolysis. Glycoside hydrolases catalyze their selective hydrolysis in oligosaccharides, polysaccharides, and glycoconjugates by following nonredox catalytic pathways or a net redox-neutral catalytic pathway using NAD(+) and divalent metal ions as cofactors. GlvA (6-phospho-alpha-glucosidase) is a glycosidase belonging to family GH4 and follows a regioselective redox-neutral mechanism of glycosidic-bond hydrolysis that favors alpha- over beta-glycosides. Its proposed catalytic mechanism can be divided into two half-reactions: the first one activates the glucopyranose ring by successively forming intermediates that are oxidized at the 3-, 2-, and 1-positions of the ring, which ultimately facilitate the heterolytic deglycosylation. The second half-reaction is essentially the reverse of the first half-reaction, beginning with the pyranose ring hydroxylation at the anomeric carbon, and it is followed by 3-reduction and regeneration of the active forms of the catalytic site and its cofactors. We investigated the NAD(+)-dependent redox mechanism of glycosidic bond hydrolysis as catalyzed by GlvA through the combined application of density functional theory and a self-consistent reaction field to a large active-site model obtained from the crystallographic structure of the enzyme, then we applied natural bond orbital and second-order perturbation analyses to monitor the electron flow and change in oxidation state on each atomic center along the reaction coordinate to rationalize the energetics and regioselectivity of this catalytic mechanism. We find that in GlvA, the redox catalytic mechanism of hydrolysis is driven by the gradual strengthening of the axial endo-anomeric component within the hexose ring along the reaction coordinate to facilitate the heterolytic dissociation of the axial C1-O bond. In addition, the combined influence of specific components of the generalized anomeric effect fully explains the regioselectivity observed in the catalytic activity of GlvA.

Spectral Alteration and Degradation of Cyanidin-3-glucoside Exposed to Pulsed Electric Field

Anthocyanins are polyphenol antioxidants that have been shown to prevent many chronic diseases. The compounds are not stable, so they tend to be decolorized or degraded during processing and storage. In this study, the spectral characteristics alteration and degradation products of cyanidin-3-glucoside (Cyd-3-glc) exposed to pulsed electric field (PEF) were investigated, and the reaction kinetics was discussed. The intensity of the UV-vis spectra decreased noticeably upon PEF treatment without modification of the spectral pattern. Protocatechuic acid and 2,4,6-trihydroxybenzoic acid were identified as degradation products of both PEF and thermally treated Cyd-3-glc, but cyanidin present in thermally treated Cyd-3-glc was absent in PEF-treated Cyd-3-glc, indicating that the first step of Cyd-3-glc degradation induced by PEF was not the hydrolysis of glycosidic bonds, which was different from that of thermal degradation. With increased electric field intensity or treatment time, the degradation of Cyd-3-glc and the formation of protocatechuic acid were enhanced; their kinetics (except 7 kV/cm for protocatechuic acid formation) were well fitted to a first-order reaction. Meanwhile, a good correlation was present between Cyd-3-glc degradation and protocatechuic acid formation.

High Speed Atomic Force Microscopy Visualizes Processive Movement of Trichoderma reesei Cellobiohydrolase I on Crystalline Cellulose

Fungal cellobiohydrolases act at liquid-solid interfaces. They have the ability to hydrolyze cellulose chains of a crystalline substrate because of their two-domain structure, i.e. cellulose-binding domain and catalytic domain, and unique active site architecture. However, the details of the action of the two domains on crystalline cellulose are still unclear. Here, we present real time observations of Trichoderma reesei (Tr) cellobiohydrolase I (Cel7A) molecules sliding on crystalline cellulose, obtained with a high speed atomic force microscope. The average velocity of the sliding movement on crystalline cellulose was 3.5 nm/s, and interestingly, the catalytic domain without the cellulose-binding domain moved with a velocity similar to that of the intact TrCel7A enzyme. However, no sliding of a catalytically inactive enzyme (mutant E212Q) or a variant lacking tryptophan at the entrance of the active site tunnel (mutant W40A) could be detected. This indicates that, besides the hydrolysis of glycosidic bonds, the loading of a cellulose chain into the active site tunnel is also essential for the enzyme movement.

Enrichment of glycopeptides for glycan structure and attachment site identification

We present a method to enrich for glycoproteins from proteomic samples. Sialylated glycoproteins were selectively periodate-oxidized, captured on hydrazide beads, trypsinized and released by acid hydrolysis of sialic acid glycosidic bonds. Mass spectrometric fragment analysis allowed identification of glycan structures, and additional fragmentation of deglycosylated ions yielded peptide sequence information, which allowed glycan attachment site and protein identification. We identified 36 N-linked and 44 O-linked glycosylation sites on glycoproteins from human cerebrospinal fluid.

Alpha-Glucosidase Promotes Hemozoin Formation in a Blood-Sucking Bug: An Evolutionary History

BackgroundHematophagous insects digest large amounts of host hemoglobin and release heme inside their guts. In Rhodnius prolixus, hemoglobin-derived heme is detoxified by biomineralization, forming hemozoin (Hz). Recently, the involvement of the R. prolixus perimicrovillar membranes in Hz formation was demonstrated.Methodology/Principal FindingsHz formation activity of an α-glucosidase was investigated. Hz formation was inhibited by specific α-glucosidase inhibitors. Moreover, Hz formation was sensitive to inhibition by Diethypyrocarbonate, suggesting a critical role of histidine residues in enzyme activity. Additionally, a polyclonal antibody raised against a phytophagous insect α-glucosidase was able to inhibit Hz formation. The α-glucosidase inhibitors have had no effects when used 10 h after the start of reaction, suggesting that α-glucosidase should act in the nucleation step of Hz formation. Hz formation was seen to be dependent on the substrate-binding site of enzyme, in a way that maltose, an enzyme substrate, blocks such activity. dsRNA, constructed using the sequence of α-glucosidase gene, was injected into R. prolixus females' hemocoel. Gene silencing was accomplished by reduction of both α-glucosidase and Hz formation activities. Insects were fed on plasma or hemin-enriched plasma and gene expression and activity of α-glucosidase were higher in the plasma plus hemin-fed insects. The deduced amino acid sequence of α-glucosidase shows a high similarity to the insect α-glucosidases, with critical histidine and aspartic residues conserved among the enzymes.Conclusions/SignificanceHerein the Hz formation is shown to be associated to an α-glucosidase, the biochemical marker from Hemipteran perimicrovillar membranes. Usually, these enzymes catalyze the hydrolysis of glycosidic bond. The results strongly suggest that α-glucosidase is responsible for Hz nucleation in the R. prolixus midgut, indicating that the plasticity of this enzyme may play an important role in conferring fitness to hemipteran hematophagy, for instance.