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Abstract: U24 is a C-terminal membrane-anchored protein found in both human herpes virus type 6 and 7 (HHV-6 and HHV-7), with an N-terminal segment that is rich in prolines (PPxY [...]

A well-balanced human diet includes a significant intake of non-starch polysaccharides, collectively termed 'dietary fibre', from the cell walls of diverse fruits and vegetables (1). Owing to the paucity of alimentary enzymes encoded by the human genome (2), our ability to derive energy from dietary fibre depends on the saccharification and fermentation of complex carbohydrates by the massive microbial community residing in our distal gut (3,4). The xyloglucans (XyGs) are a ubiquitous family of highly branched plant cell wall polysaccharides (5,6) whose mechanism(s) of degradation in the human gut and consequent importance in nutrition have been unclear (1,7,8). Here we demonstrate that a single, complex gene locus in Bacteroides ovatus confers XyG catabolism in this common colonic symbiont. Through targeted gene disruption, biochemical analysis of all predicted glycoside hydrolases and carbohydrate-binding proteins, and three-dimensional structural determination of the vanguard endo-xyloglucanase, we reveal the molecular mechanisms through which XyGs are hydrolysed to component monosaccharides for further metabolism. We also observe that orthologous XyG utilization loci (XyGULs) serve as genetic markers of XyG catabolism in Bacteroidetes, that XyGULs are restricted to a limited number of phylogenetically diverse strains, and that XyGULs are ubiquitous in surveyed human metagenomes. Our findings reveal that the metabolism of even highly abundant components of dietary fibre maybe mediated by niche species, which has immediate fundamental and practical implications for gut symbiont population ecology in the context of human diet, nutrition and health (9-12)., Despite our omnivory, a census of the glycoside hydrolases (GHs) encoded by the human genome indicates that our inherent ability to digest carbohydrates is restricted to starch and simple saccharides [...]

Results demonstrate that C-terminal family carbohydrate-binding module of xylanase binds to all forms of cellulose and binds specifically to the reducing ends of the soluble polysaccharides and cellulose. Data indicate that the binding is specific to two glucose units of the carbohydrates.

Author(s): Yu Luo [1, 2]; Elizabeth A. Frey [1, 2]; Richard A. Pfuetzner [1, 2]; A. Louise Creagh [2]; Derek G. Knoechel [1, 2]; Charles A. Haynes [2]; B. Brett [...]

Research has been conducted on the Cellulomonas fimi 1,4-beta-glucanase CenC cellulose-binding domains. The tertiary structure of these domains has been investigated via heteronuclear magnetic resonance spectroscopy.

A study was conducted to examine the interactions of the N-terminal cellulose-binding domain from Cellulomonas fimi beta-1,4-glucanase CenC with calcium by calorimetry and nuclear magnetic resonance (NMR) spectroscopy. A 50 mM sodium chloride was utilized as a buffer for the NMR studies while a line shape analysis was carried out to determine calcium kinetics. Results indicated that metal ion binding was thermodynamically correlated with native-state stability.

A study was conducted to analyze the thermal stability and oligosaccharide-binding thermodynamics of the N-terminal cellulose-binding domain of the Cellulomonas fimi endoglucanase cenC. Differential scanning calorimetry was utilized to measure stability and oligosaccharide-binding thermodynamics. Results revealed that the intramolecular link in the Cellulose fimi gene contributed to the stability of the endoglucanase cenC and a global two-dimensional minimization routine was utilized to regress the unfolding thermodynamics for the endoglucanase cenC-cellopentaose system.

The activity of the beta-1,4-glycanase Cex from Cellulomonas fimi was studied in connection with its industrial application in cellulose hydrolysis and its possible utilization in cellosaccharide synthesis. Findings indicated that the enzyme is irreversibly deactivated in one of two possible ways: through a low-temperature route characterized by first-order kinetics or through a high-temperature route characterized by an initial reversible step followed by an irreversible step.

The carbohydrate-binding specificity of the N-terminal cellulose-binding domain (CBDN1) from Cellulomonas fimi beta-1,4-glucanase C (CenC) was studied utilizing affinity electrophoresis, binding assays and microcalorimetry in parallel with NMR and difference ultraviolet absorbance spectroscopy. CBDN1 binds amorphous cellulose with high affinity, binds Avicel weakly and does not bind highly crystalline bacterial or tunicin cellulose. Also, CBDN1 binds soluble cellooligosaccharides and beta-1,4-linked oligomers of glucose such as hydroxyethylcellulose, soluble beta-1,3-1,5-glucans from barley and oat, but has no affinity for alpha-1,4-, beta-1,3, or beta-1,6-polymers of glucose.

ABSTRACT Isoelectric chromatofocusing (ICF), a mode of chromatography by which proteins are separated based on changes in their charge state with pH, is widely used at analytical scales and finding increasing interest in biologics manufacturing due to its exceptional resolving power. Here, a method is described for using simple monoprotic and diprotic buffers to create stable mobile phases for sample loading on a strong anion exchange column and for achieving an elution pH gradient of desired shape covering any pH range from pH 10.0 to 3. The buffers used are selected to satisfy cost constraints, and to permit facile detection of eluted biologics by UV spectroscopy and mass spectrometry. The method exploits a new model described here that combines multiple-chemical and adsorption-equilibria theory to enable in silico tailoring of elution pH profiles using mixtures of these simple buffers. It is shown to provide a versatile platform for optimizing and conducting ICF of protein mixtures on strong anion exchange media. Biotechnol. Bioeng. 2014;111: 552-564. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

We performed quantitative analysis of the binding kinetics and affinity of carbohydrate--lectin binding and correlated them directly with the molecular and structural features of ligands presented at the nanoscale within the glycocalyx mimicking layers on surfaces. The surface plasmon resonance analysis identified that the mode of binding changed from multivalent to monovalent, which resulted in a near 1000-fold change in the equilibrium association constant, by varying the spatial distribution of carbohydrate ligands within the surface-grafted polymer layer. We identified, for the first time, that the manner in which the ligands presented on the surface has great influence on the binding at the first stage of bivalent chelating, not on the binding at the second stage. The rupture forces measured by atomic force microscope force spectroscopy also indicated that the mode of binding between lectin and ligands changed from multiple to single with variation in the ligand presentation. The dependence of lectin binding on the glycopolymer composition and grafting density is directly correlated with the nanoscale presentation of ligands on a surface, which is a determining factor in controlling the clustering and statistical effects contributing to the enhanced binding [ABSTRACT FROM AUTHOR]

The intrinsic antimicrobial resistance of the opportunistic human pathogen Pseudomonas aeruginosa is compounded in mutant strains that overexpress multidrug efflux pumps such as the prominent drug-proton antiporter, MexAB-OprM. The primary regulator of the mexAB-oprM operon is the MarR family repressor, MexR. An additional repressor, NalC, also regulates mexAB-oprM by controlling expression of ArmR, an antirepressor peptide that is hypothesized to prevent the binding of MexR to its cognate DNA operator via an allosteric protein-peptide interaction. To better understand how ArmR modulates MexR, we determined the MexRbinding region of ArmR as its C-terminal 25 residues and solved the crystal structure of MexR in a 2:1 complex with this ArmR fragment at 1.8 Å resolution. This structure reveals that the C-terminal residues of ArmR form a kinked a-helix, which occupies a pseudosymmetrical and largely hydrophobic binding cavity located at the centre of the MexR dimer. Although the ArmR-binding cavity partially overlaps with the small molecule effector-binding sites of other MarR family members, it possesses a larger and more complex binding surface to accommodate the greater size and specific physicochemical properties of a peptide effector. Comparison with the structure of apo-MexR reveals that ArmR stabilizes a dramatic conformational change that is incompatible with DNAbinding. Thus, this work defines the structural mechanism by which ArmR allosterically derepresses MexR-controlled gene expression in P. aeruginosa and reveals important insights into the regulation of multidrug resistance. [ABSTRACT FROM AUTHOR]

Examines the interaction of polysaccharides with the N-terminal cellulose-binding domain (CBD) of `Cellulomonas fimi' CenC. Comparison of CBC with other cellulose-binding domains; Investigation of the thermodynamics of the binding of CBD to oligosaccharides, soluble glycans and phosphoric acid-swollen cellulose; Contribution of electrostatic effects.