Promiscuous Yet Specific: A Methionine-Aromatic Interaction Drives the Reaction Scope of the Family 1 Glycosyltransferase UGT88E3 from Soybean.

Family 1 glycosyltransferases (GT1s, UGTs) catalyze the regioselective glycosylation of natural products in a single step. We identified UGT88E3 as a particularly promising biocatalyst able to produce a variety of pure, single glycosidic products from polyphenols with high chemical yields. We investigated this particularly desirable duality toward specificity, i.

Structural elucidation of novel pro-inflammatory polysaccharides from Daphne mezereum L.

Daphne mezereum L., an important medicinal plant in Scandinavian folk medicine, was used to treat ailments such as diarrhea, swelling and stomach pain. A range of natural compounds have been isolated, but little attention has been given to the polysaccharides in this plant.

The Engineered Hexosaminidase OGA-D120N Is an Efficient -/-/-Glycoligase That Also Catalyzes Formation and Release of Oxazoline Donors for Cascade Syntheses with Glycosynthases or Transglycosylases.

Engineering glycoside hydrolases is a major route to obtaining catalysts forming glycosidic bonds. Glycosynthases, thioglycoligases, and transglycosylases represent the main strategies, each having advantages and drawbacks. Here, we show that an engineered enzyme from the GH84 family, the acid-base mutant OGA-D120N, is an efficient -, -, and -glycoligase, able to use , , , and nucleophiles.

Family 1 Glycosyltransferase UGT706F8 from Selectively Catalyzes the Synthesis of Silibinin 7--β-d-Glucoside.

Regioselective glycosylation is a chemical challenge, leading to multistep syntheses with protecting group manipulations, ultimately resulting in poor atom economy and compromised sustainability. Enzymes allow eco-friendly and regioselective bond formation with fully deprotected substrates in a single reaction. For the selective glucosylation of silibinin, a pharmaceutical challenged with low solubility, enzyme engineering has previously been employed, but the resulting yields and were limited, prohibiting the application of the engineered catalyst.

Full NMR assignment, revised structure and biosynthetic analysis for the capsular polysaccharide from Streptococcus Pneumoniae serotype 15F.

Upon investigation of Streptococcus pneumoniae serotype 15F capsular polysaccharide (CPS), we discovered that it had a different phosphorylation substituent, namely glycerol-2-phosphate like the other serogroup 15 CPS rather than the originally reported 0.2 equivalent of phosphate or phosphocholine. Furthermore, we also determined the locations of the two previously unassigned O-acetyl groups present in the repeating unit of the 15F CPS, and carried out full NMR assignments of the 15F as well as 15A CPS.

Rational Enzyme Design without Structural Knowledge: A Sequence-Based Approach for Efficient Generation of Transglycosylases.

Glycobiology is dogged by the relative scarcity of synthetic, defined oligosaccharides. Enzyme-catalysed glycosylation using glycoside hydrolases is feasible but is hampered by the innate hydrolytic activity of these enzymes. Protein engineering is useful to remedy this, but it usually requires prior structural knowledge of the target enzyme, and/or relies on extensive, time-consuming screening and analysis.

Structural, Genetic, and Serological Elucidation of Streptococcus pneumoniae Serogroup 24 Serotypes: Discovery of a New Serotype, 24C, with a Variable Capsule Structure.

Pneumococcal capsules are important in pneumococcal pathogenesis and vaccine development. Although conjugate vaccines have brought about a significant reduction in invasive pneumococcal disease (IPD) caused by vaccine serotypes, the relative serotype prevalence has shifted with the dramatic emergence of serotype 24F in some countries. Here, we describe 14 isolates (13 IPD and 1 non-IPD) expressing a new capsule type, 24C, which resembles 24F but has a novel serological profile.

Binding Sites for Oligosaccharide Repeats from Lactic Acid Bacteria Exopolysaccharides on Bovine β-Lactoglobulin Identified by NMR Spectroscopy.

Lactic acid bacterial exopolysaccharides (EPS) are used in the food industry to improve the stability and rheological properties of fermented dairy products. β-Lactoglobulin (BLG), the dominant whey protein in bovine milk, is well known to bind small molecules such as fatty acids, vitamins, and flavors, and to interact with neutral and anionic polysaccharides used in food and pharmaceuticals. While sparse data are available on the affinity of EPS-milk protein interactions, structural information on BLG-EPS complexes, including the EPS binding sites, is completely lacking.

Structural, biosynthetic and serological cross-reactive elucidation of capsular polysaccharides from Streptococcus pneumoniae serogroup 28.

Capsular polysaccharides (CPS) are the key virulent factors in the pathogenesis of Streptococcus pneumoniae. The previously unknown CPS structures of the pneumococcal serotype 28F and 28A were thoroughly characterized by NMR spectroscopy, chemical analysis and AF4-MALS-dRI. The following repeat unit structures were determined: -4)[α-l-Rhap-[4-P-2-Gro]]-(1-3)-α-d-Sug-[6-P-Cho]-(1-3)-β-l-Rhap-[2-OAc]-(1-4)-β-d-Glcp-(1-; 28F: Sug = Glcp, Mw: 540.

Bilirubin oxidase oriented on novel type three-dimensional biocathodes with reduced graphene aggregation for biocathode.

Aggregation of reduced graphene oxide (RGO) due to π-π stacking is a recurrent problem in graphene-based electrochemistry, decreasing the effective working area and therefore the performance of the RGO electrodes. Dispersing RGO on three-dimensional (3D) carbon paper electrodes is one strategy towards overcoming this challenge, with partial relief aggregation. In this report, we describe the grafting of negatively charged 4-aminobenzoic acid (4-ABA) onto a graphene functionalized carbon paper electrode surface.

Unexpected Anomeric Acceptor Preference Observed Using dDNP NMR for Transglycosylation Studies of β-Galactosidases.

The transglycosylation abilities of β-galactosidases were investigated using hyperpolarized [U-C,U-H]glucose as an acceptor and -nitrophenyl β-galactopyranoside as a donor. Several products were readily observable, and at least in the case when O3 acted as an acceptor, the enzymes showed a clear selectivity toward the β-anomer of glucose. Additionally, it was possible to determine the relative hydrolysis rates of the formed transglycosylation products, providing information on the selectivity as well.

Identification and Characterization of a β--Acetylhexosaminidase with a Biosynthetic Activity from the Marine Bacterium S66.

β--Acetylhexosaminidases are glycoside hydrolases (GHs) acting on -acetylated carbohydrates and glycoproteins with the release of -acetylhexosamines. Members of the family GH20 have been reported to catalyze the transfer of -acetylglucosamine (GlcNAc) to an acceptor, i.e.

Alginate Trisaccharide Binding Sites on the Surface of β-Lactoglobulin Identified by NMR Spectroscopy: Implications for Molecular Network Formation.

β-lactoglobulin (BLG) is a promiscuous protein in terms of ligand interactions, having several binding sites reported for hydrophobic biomolecules such as fatty acids, lipids, and vitamins as well as detergents. BLG also interacts with neutral and anionic oligo- and polysaccharides for which the binding sites remain to be identified. The multivalency offered by these carbohydrate ligands is expected to facilitate coacervation, an electrostatically driven liquid-liquid phase separation.

Structural, Biosynthetic, and Serological Cross-Reactive Elucidation of Capsular Polysaccharides from Streptococcus pneumoniae Serogroup 16.

Capsular polysaccharides (CPS) are crucial virulence factors of The previously unknown CPS structures of the pneumococcal serogroup 16 (serotypes 16F and 16A) were thoroughly elucidated by nuclear magnetic resonance (NMR) spectroscopy and verified by chemical analysis. The following repeat unit structures were determined: 16F, -3)-α-l-Rha-[4-P-1-Gro]-(1-3)-α-d-Glc-[(6-P-1)-Gro]-(1-3)-β-l-Rha-[2-OAc]-(1-4)-β-d-Glc-(1-; 16A, -3)-β-d-Gal-[2-OAc (70%)]-(1-3)-α-l-Rha-(1-2)-α-l-Rha-(1-3)-α-d-Gal-[(6-P-1)-Gro]-(1-3)-β-d-Gal-(1-4)-β-d-Glc-(1- (OAc, O-acetyl substitution; P-1-Gro, glycerol-1-phosphate substitution) A further analysis of CPS biosynthesis of serotypes 16F and 16A, in conjunction with published gene bioinformatics analysis and structures of related serotypes, revealed presumable specific function of glycosyltransferase, acetyltransferase, phosphotransferase, and polymerase. The functions of glycosyltransferases WcxN and WcxT were proposed for the first time, and they were assigned to catalyze linkage of α-l-Rha-(1-3)-α-d-Glc and α-l-Rha-(1-2)-α-l-Rha, respectively.

Discovery and description of a new serogroup 7 Streptococcus pneumoniae serotype, 7D, and structural analysis of 7C and 7D.

Streptococcus pneumoniae is characterised into 92 serotypes based on antigenic reactions of commercial rabbit sera to the capsular polysaccharides. During development of a bioinformatic serotyping tool (PneumoCaT), an isolate exhibited a novel codon at residue 385 of the glycosyltransferase gene wcwK encoding a distinct amino acid, which differentiates genogroup 7. Investigation by repeat serotyping and Quellung reaction revealed a novel pattern of factor sera with the isolate reacting very strongly with 7f, but also with 7e factor sera.

Discovery of Intermediates of lacZ β-Galactosidase Catalyzed Hydrolysis Using dDNP NMR.

Using dissolution dynamic nuclear polarization, the sensitivity of single scan solution state C NMR can be improved up to 4 orders of magnitude. In this study, the enzyme lacZ β-galactosidase from Escherichia coli was subjected to hyperpolarized substrate, and previously unknown reaction intermediates were observed, including a 1,1-linked disaccharide. The enzyme is known for making 1,6-transglycosylation, producing products like allolactose, that are also substrates.

Structural characterization of bioactive heteropolysaccharides from the medicinal fungus Inonotus obliquus (Chaga).

The aim of this paper was to perform a comprehensive characterization of polysaccharides isolated from the interior (IOI) and exterior (IOE) parts of the fungus Inonotus obliquus. Pre-extraction with DCM and MeOH, followed by water and alkali extraction and ethanol precipitation gave two water extracts and two alkali extracts. Neutral and acidic polysaccharide fractions were obtained after anion-exchange chromatography of the water extracts.

Chemodiversity of Ladder-Frame Prymnesin Polyethers in Prymnesium parvum.

Blooms of the microalga Prymnesium parvum cause devastating fish kills worldwide, which are suspected to be caused by the supersized ladder-frame polyether toxins prymnesin-1 and -2. These toxins have, however, only been detected from P. parvum in rare cases since they were originally described two decades ago.

A diverse range of bacterial and eukaryotic chitinases hydrolyzes the LacNAc (Galβ1-4GlcNAc) and LacdiNAc (GalNAcβ1-4GlcNAc) motifs found on vertebrate and insect cells.

There is emerging evidence that chitinases have additional functions beyond degrading environmental chitin, such as involvement in innate and acquired immune responses, tissue remodeling, fibrosis, and serving as virulence factors of bacterial pathogens. We have recently shown that both the human chitotriosidase and a chitinase from Salmonella enterica serovar Typhimurium hydrolyze LacNAc from Galβ1-4GlcNAcβ-tetramethylrhodamine (LacNAc-TMR (Galβ1-4GlcNAcβ(CH2)8CONH(CH2)2NHCO-TMR)), a fluorescently labeled model substrate for glycans found in mammals. In this study we have examined the binding affinities of the Salmonella chitinase by carbohydrate microarray screening and found that it binds to a range of compounds, including five that contain LacNAc structures.

In vitro growth of four individual human gut bacteria on oligosaccharides produced by chemoenzymatic synthesis.

The present study aimed at examining oligosaccharides (OS) for potential stimulation of probiotic bacteria. Nineteen structurally well-defined candidate OS covering groups of β-glucosides, α-glucosides and α-galactosides with degree of polymerization 2-4 were prepared in >100 mg amounts by chemoenzymatic synthesis (i.e.

NMR assignment of structural motifs in intact β-limit dextrin and its α-amylase degradation products in situ.

An increasingly detailed and realistic view of biological processes often hinges on atomic-level characterization of biomacromolecules and of the processes they are involved in, preferably under near-physiological conditions. Structure, degradation, and synthesis of glucose storage polymers have been studied for decades with a range of analytical tools, but the detailed in situ analysis has remained an analytical challenge. Here, we report the NMR assignment of different structural motifs in the β-limit dextrin from lintnerized maize starch as a branched α-glucan model system for starch, which is depleted of repetitive α-(1→4) glycosidic bonds at non-reducing ends but has the α-(1→6) branch points intact.

Chemical and biological characterization of pectin-like polysaccharides from the bark of the Malian medicinal tree Cola cordifolia.

The bark of Cola cordifolia used in Malian traditional medicine contains unusual types of polysaccharides with immunomodulating activities. We report for the first time on the structure of a polymer designated CC1P1 having the repeating structure [2→)[α-D-Gal(1→3)]α-L-Rha(1→4)α-d-GalA(1→] as determined by NMR and GC/MS. α-Linked Gal is unusual in pectins.

Atlantinone A, a Meroterpenoid Produced by Penicillium ribeum and Several Cheese Associated Penicillium Species.

Atlantinone A has been isolated from the psychrotolerant fungus Penicillium ribeum. The exact structure of the compound was confirmed by mass spectrometric and 1- and 2D NMR experiments. Atlantinone A was originally only produced upon chemical epigenetic manipulation of P.

Real-time detection of central carbon metabolism in living Escherichia coli and its response to perturbations.

The direct tracking of cellular reactions in vivo has been facilitated with recent technologies that strongly enhance NMR signals in substrates of interest. This methodology can be used to assay intracellular reactions that occur within seconds to few minutes, as the NMR signal enhancement typically fades on this time scale. Here, we show that the enhancement of (13)C nuclear spin polarization in deuterated glucose allows to directly follow the flux of glucose signal through rather extended reaction networks of central carbon metabolism in living Escherichia coli.