Differential Dynamics at Glycosidic Linkages of an Oligosaccharide as Revealed by 13C NMR Spin Relaxation and Stochastic Modeling

Among biomolecules, carbohydrates are unique in that not only can linkages be formed through different positions but the structures may also be branched. The trisaccharide \uf062-D-Glcp-(1\uf0ae3)[\uf062-D-Glcp-(1\uf0ae2)]-\uf061-D-Manp-OMe represents a model of a branched vicinally disubstituted structure. A 13C site-specific isotopologue with labeling in each of the two terminal glucosyl residues enabled acquisition of high-quality 13C NMR relaxation parameters T1, T2 and heteronuclear NOE, with standard deviations of \uf0a3 0.5%. For interpretation of the experimental NMR data a diffusive chain model was used in which the dynamics of the glycosidic linkages is coupled to the global reorientation motion of the trisaccharide. Brownian dynamics simulations relying on the potential of mean force at the glycosidic linkages were employed to evaluate spectral densities of the spin probes. Calculated NMR relaxation parameters showed very good agreement with experimental data, deviating < 3%. The resulting dynamics is described by correlation times of 196 ps and 174 ps for the \uf062-(1\uf0ae2)- and \uf062-(1\uf0ae3)-linked glucosyl residues, respectively, i.e., different and linkage dependent. Notably, the devised computational protocol was performed without any fitting of parameters

Serotype-conversion in Shigella flexneri: identification of a novel bacteriophage, Sf101, from a serotype 7a strain

BACKGROUND Shigella flexneri is the major cause of bacillary dysentery in the developing countries. The lipopolysaccharide (LPS) O-antigen of S. flexneri plays an important role in its pathogenesis and also divides S. flexneri into 19 serotypes. All the serotypes with an exception for serotype 6 share a common O-antigen backbone comprising of N-acetylglucosamine and three rhamnose residues. Different serotypes result from modification of the basic backbone conferred by phage-encoded glucosyltransferase and/or acetyltransferase genes, or plasmid-encoded phosphoethanolamine transferase. Recently, a new site for O-acetylation at positions 3 and 4 of RhaIII, in serotypes 1a, 1b, 2a, 5a and Y was shown to be mediated by the oacB gene. Additionally, this gene was shown to be carried by a transposon-like structure inserted upstream of the adrA region on the chromosome. RESULTS In this study, a novel bacteriophage Sf101, encoding the oacB gene was isolated and characterised from a serotype 7a strain. The complete sequence of its 38,742 bp genome encoding 66 open reading frames (orfs) was determined. Comparative analysis revealed that phage Sf101 has a mosaic genome, and most of its proteins were >90% identical to the proteins from 12 previously characterised lambdoid phages. In addition, the organisation of Sf101 genes was found to be highly similar to bacteriophage Sf6. Analysis of the Sf101 OacB identified two amino acid substitutions in the protein; however, results obtained by NMR spectroscopy confirmed that Sf101-OacB was functional. Inspection of the chromosomal integration site of Sf101 phage revealed that this phage integrates in the sbcB locus, thus unveiling a new site for integration of serotype-converting phages of S. flexneri, and determining an alternative location of oacB gene in the chromosome. Furthermore, this study identified oacB gene in several serotype 7a isolates from various regions providing evidence of O-acetyl modification in serotype 7a. CONCLUSIONS This is the first report on the isolation of bacteriophage Sf101 which contains the S. flexneri O-antigen modification gene oacB. Sf101 has a highly mosaic genome and was found to integrate in the sbcB locus. These findings contribute an advance in our current knowledge of serotype converting phages of S. flexneri.This work was supported by grants from the Swedish Research Council and The Knut and Alice Wallenberg Foundation to GW

flexibility at a glycosidic linkage revealed by molecular dynamics stochastic modeling and 13c nmr spin relaxation conformational preferences of α l rhap α 1 2 α l rhap ome in water and dimethyl sulfoxide solutions

Coupling between global reorientation and local motion is essential for reproducing NMR relaxation parameters of a flexible molecule

A detailed picture of a protein–carbohydrate hydrogen-bonding network revealed by NMR and MD simulations

Cyanovirin-N (CV-N) is a cyanobacterial lectin with antiviral activity towards HIV and several other viruses. Here, we identify mannoside hydroxyl protons that are hydrogen bonded to the protein backbone of the CV-N domain B binding site, using NMR spectroscopy. For the two carbohydrate ligands Manα(1→2)ManαOMe and Manα(1→2) Manα(1→6)ManαOMe five hydroxyl protons are involved in hydrogen-bonding networks. Comparison with previous crystallographic results revealed that four of these hydroxyl protons donate hydrogen bonds to protein backbone carbonyl oxygens in solution and in the crystal. Hydrogen bonds were not detected between the side chains of Glu41 and Arg76 with sugar hydroxyls, as previously proposed for CV-N binding of mannosides. Molecular dynamics simulations of the CV-N/Manα(1→2)Manα(1→6)ManαOMe complex confirmed the NMR-determined hydrogen-bonding network. Detailed characterization of CV-N/mannoside complexes provides a better understanding of lectin-carbohydrate interactions and opens up to the use of CV-N and similar lectins as antiviral agents

An OregonGreen488-labelled d-amino acid for visualizing peptidoglycan by super-resolution STED nanoscopy

Fluorescent d-amino acids (FDAAs) are molecular probes that are widely used for labelling the peptidoglycan layer of bacteria. When added to growing cells they are incorporated into the stem peptide by a transpeptidase reaction, allowing the timing and localization of peptidoglycan synthesis to be determined by fluorescence microscopy. Herein we describe the chemical synthesis of an OregonGreen488-labelled FDAA (OGDA). We also demonstrate that OGDA can be efficiently incorporated into the PG of Gram-positive and some Gram-negative bacteria, and imaged by super-resolution stimulated emission depletion (STED) nanoscopy at a resolution well below 100 nm

Ethyl 3,6-di-O-benzyl-2-de­oxy-N-phthalimido-1-thio-β-d-glucopyran­oside

In the title compound, C30H31NO6S, the plane of the N-phthalimido group is nearly orthogonal to the least-squares plane of the sugar ring (defined by atoms C2, C3, C5 and O5 using standard glucose nomenclature), making a dihedral angle of 72.8 (1)°. The thio­ethyl group has the exo-anomeric conformation. The hy­droxy group forms an inter­molecular hydrogen bond to the O atom in the sugar ring, generating [100] chains. There are four close π–π contacts with centroid–centroid distances less than 4.0 Å, all with dihedral angles between the inter­acting π systems of only ≃ 8°, supporting energetically favourable stacking inter­actions

Methyl 3-O-α-d-mannopyranosyl β-d-glucopyran­oside tetra­hydrate

The title compound, C13H24O11·4H2O, forms extended hydrogen-bonded networks. These are present between disaccharides, but not as inter-residue hydrogen bonds, as well as to water mol­ecules that in addition form an inter­molecular chain of hydrogen bonds. The conformation of the disaccharide is described by the glycosidic torsion angles ϕH = −34° and ψH = −5°. Macroscopically, the disaccharide was observed to be hygroscopic

(Z)-1,2:5,6-Di-O-isopropyl­idene-α-d-ribo-hexofuranos-3-ulose O-benzyl­oxime

The title compound, C19H25NO6, is a Z diastereomer in which the phenyl ring of the 3-benzyl­oxime substituent and the 5,6-O-isopropyl­idene acetal are both located on the Si-face of the C=N double bond. Inter­molecular C—H⋯O inter­actions result in helical chains along the b axis of the monoclinic unit cell

Tautomers of N-acetyl-d-allosamine: an NMR and computational chemistry study

d-Allosamine is a rare sugar in Nature but its pyranoid form has been found alpha-linked in the core region of the lipopolysaccharide from the Gram-negative bacterium Porphyromonas gingivalis and in the chitanase inhibitor allosamidin, then beta-linked and N-acetylated. In water solution the monosaccharide N-acetyl-d-allosamine (d-AllNAc) shows a significant presence of four tautomers arising from pyranoid and furanoid ring forms and anomeric configurations. The furanoid ring forms both showed (3)J(H1,H2) approximate to 4.85 Hz and to differentiate the anomeric configurations a series of chemical shift anisotropy/dipole-dipole cross-correlated relaxation NMR experiments was performed in which the alpha-anomeric form showed notable different relaxation rates for its components of the H1 doublet, thereby making it possible to elucidate the anomeric configuration of each of the furanoses. The conformational preferences of the different forms of d-AllNAc were investigated by (3)J(HH), (2)J(CH) and (3)J(CH) coupling constants from NMR experiments, molecular dynamics simulations and density functional theory calculations. The pyranose form resides in the C-4(1) conformation and the furanose ring form has the majority of its conformers located on the South-East region of the pseudorotation wheel, with a small population in the Northern hemisphere. The tautomeric equilibrium was quite sensitive to changes in temperature, where the beta-anomer of the pyranoid ring form decreased upon a temperature increase while the other forms increased

Structural characterization of an all-aminosugar-containing capsular polysaccharide from Colwellia psychrerythraea 34H

Colwellia psychrerythraea strain 34H, a Gram-negative bacterium isolated from Arctic marine sediments, is considered a model to study the adaptation to cold environments. Recently, we demonstrated that C. psychrerythraea 34H produces two different extracellular polysaccharides, a capsular polysaccharide and a medium released polysaccharide, which confer cryoprotection to the bacterium. In this study, we report the structure of an additional capsular polysaccharide produced by Colwellia grown at a different temperature. The structure was determined using chemical methods, and one- and two-dimensional NMR spectroscopy. The results showed a trisaccharide repeating unit made up of only amino-sugar residues: N-acetyl-galactosamine, 2,4-diacetamido-2,4,6-trideoxy-glucose (bacillosamine), and 2-acetamido-2-deoxyglucuronic acid with the following structure: →4)-β-d-GlcpNAcA-(1 →3)-β-d-QuipNAc4NAc-(1 →3)-β-d-GalpNAc-(1 →. The 3D model, generated in accordance with 1H,1H-NOE NMR correlations and consisting of ten repeating units, shows a helical structure. In contrast with the other extracellular polysaccharides produced from Colwellia at 4 °C, this molecule displays only a low ice recrystallization inhibition activity