Aldobiouronic Acid Research Articles

Structural characterization and osteogenic bioactivity of a sulfated polysaccharide from pacific abalone (Haliotis discus hannai Ino)

Bone morphogenic protein-2 (BMP-2) is known to promote osteogenesis. To find novel adjuvants to enhance the activity of BMP-2, the present study investigated the structure BMP-2-induced osteogenic activity of a water-soluble polysaccharide from the gonad of pacific abalone (Haliotis discus hannai Ino) named AGSP. Through analysis of aldobiouronic acids released from AGSP, monosaccharide composition comparison of AGSP and its reduced product, and methylation analysis and NMR analysis of AGSP and its desulfated derivative, the main structure residue of AGSP was determined as →3)-GlcA(1→3)-Gal(1→ with sulfated branches comprised of prevelant Gal and minor Glc, and →4)-β-GlcA(1→2)-α-Man(1→ residue was also found. AGSP possessed a sulfate content of 12.4% with a relative molecular weight of 6.6kDa. AGSP strengthened alkaline phosphatase activity induced by BMP-2 in a dose dependent manner at 10–200μg/mL with 425% enhancement being observed at 200μg/mL, indicating AGSP could be an adjuvant candidate to enhance osteogenic activity of BMP-2.

Insight into glycoside hydrolases for debranched xylan degradation from extremely thermophilic bacterium Caldicellulosiruptor lactoaceticus.

Caldicellulosiruptor lactoaceticus 6A, an anaerobic and extremely thermophilic bacterium, uses natural xylan as carbon source. The encoded genes of C. lactoaceticus 6A for glycoside hydrolase (GH) provide a platform for xylan degradation. The GH family 10 xylanase (Xyn10A) and GH67 α-glucuronidase (Agu67A) from C. lactoaceticus 6A were heterologously expressed, purified and characterized. Both Xyn10A and Agu67A are predicted as intracellular enzymes as no signal peptides identified. Xyn10A and Agu67A had molecular weight of 47.0 kDa and 80.0 kDa respectively as determined by SDS-PAGE, while both appeared as homodimer when analyzed by gel filtration. Xyn10A displayed the highest activity at 80°C and pH 6.5, as 75°C and pH 6.5 for Agu67A. Xyn10A had good stability at 75°C, 80°C, and pH 4.5–8.5, respectively, and was sensitive to various metal ions and reagents. Xyn10A possessed hydrolytic activity towards xylo-oligosaccharides (XOs) and beechwood xylan. At optimum conditions, the specific activity of Xyn10A was 44.6 IU/mg with beechwood xylan as substrate, and liberated branched XOs, xylobiose, and xylose. Agu67A was active on branched XOs with methyl-glucuronic acids (MeGlcA) sub-chains, and primarily generated XOs equivalents and MeGlcA. The specific activity of Agu67A was 1.3 IU/mg with aldobiouronic acid as substrate. The synergistic action of Xyn10A and Agu67A was observed with MeGlcA branched XOs and xylan as substrates, both backbone and branched chain of substrates were degraded, and liberated xylose, xylobiose, and MeGlcA. The synergism of Xyn10A and Agu67A provided not only a thermophilic method for natural xylan degradation, but also insight into the mechanisms for xylan utilization of C. lactoaceticus.

Aldobiouronic acid domains in Helicobacter pylori

Helicobacter pylori cell-surface glycans exert strong influences in host–microbe interplays and define the strain’s immunological signature. Envisaging the development of a carbohydrate-based vaccine against the gastroduodenal pathogen H. pylori, several clinical isolates are being screened for their cell-surface glycan profile. The present work concerns H. pylori clinical specimen PTAV79 that abundantly expressed amylose-like glycans. These polysaccharides were isolated in glycan-rich fractions resultant from phenol–water extractions and purified by Bio-Gel P2. Structural studies showed that the glycans are linked to glycerol and present aldobiouronic acid domains composed of [→3)-α- d-GlcA-(1→4)-α- d-Glc-(1→] repeating units. The amylose domains were constituted by an average of 19 Glc residues and the acidic moieties had an average number of 10 aldobiouronic acid repeating units. These polysaccharides were isolated in fractions that, although hydrophilic, were rich in stearic acid, strongly suggesting that they are present as glycerolipids anchored to cell-surface.

Structural unit of xylans from sugi (Cryptomeria japonica) and hinoki (Chamaecyparis obtusa)

Arabinoglucuronoxylans (AGXs) isolated from the holocellulose of sugi (Cryptomeria japonica) and hinoki (Chamaecyparis obtusa) contained one 4-O-methyl-d-glucopyranosyluronic acid (4-O-Me-d-GlcAp) residue per 6.2 d-xylopyranose (d-Xylp) residues and one 4-O-Me-d-GlcAp residue per 3.8 d-Xylp residues. These AGXs were subjected to partial acid hydrolysis. Analyses by size exclusion chromatography and electrospray-ionization mass spectroscopy of the neutral sugar fractions in the hydrolysates showed the presence of xylooligosaccharides having a degree of polymerization of 2-8 in addition to d-Xyl, suggesting that the AGXs from sugi and hinoki contained unsubstituted chains consisting of at least eight d-Xyl residues. The acidic sugars in the hydrolysates were separated into two series of aldouronic acids composed of 4-O-Me-d-GlcAp and d-Xylp by ion-exchange chromatography. The first series included aldouronic acids from aldobiouronic acid (4-O-Me-d-GlcAp-Xyl) to aldopentaouronic acids (4-O-Me-d-GlcAp-Xyl4). The second series were aldouronic acids composed of two 4-O-Me-d-GlcAp residues and 2-4 d-Xyl residues. In these acidic sugars, the uronic acid side chains were located on two contiguous d-Xyl residues. These facts indicated that AGXs from sugi and hinoki had a structural unit containing two 4-O-Me-d-GlcAp residues on two contiguous d-Xyl residues as well as AGXs from spruce and larch.

ChemInform Abstract: Improved Synthesis of an Aldobiouronic Acid Related to Hardwood Xylans, and Preparation of a Derivative Thereof Suitable for Linking to Proteins.

Treatment of 1,3,4-tri-O-acetyl-alpha-D-xylopyranose with methyl 2,3-di-O-benzyl-l-chloro-l-deoxy-4-O-methyl-alpha, beta-D-glucopyranuronate in the presence of silver trifluoromethanesulfonate was highly stereoselective to give the alpha-linked aldobiouronic acid derivative (4) in 86% yield, after hydrogenolysis of the crude product of the coupling and chromatography. Compound 4 was acetylated and the fully protected substance was converted to the corresponding glycosyl chloride. Reaction of the latter with p-nitrophenol under phase-transfer catalysis afforded, after deacetylation, p-nitrophenyl 2-O-(methyl 4-O-methyl-alpha-D-glucopyranosyluronate)-beta-D-xylopyranoside.

Anionic derivatives of xyloglucan function as acceptor but not donor substrates for xyloglucan endotransglucosylase activity

Tamarind xyloglucan was oxidised by reaction with sodium hypochlorite in the presence of 2,2,6,6-tetramethyl-1-piperidinyloxy free radical (TEMPO). Galactose residues and non-xylosylated glucose residues were thus converted into galacturonic and glucuronic acid residues, respectively, producing an anionic polysaccharide. Acid hydrolysis of oxidised xyloglucan yielded two aldobiouronic acids, deduced to be beta-D: -GalpA-(1-->2)-D-Xyl and beta-D: -GlcpA-(1-->4)-D-Glc. Anionic xyloglucan had a decreased ability to hydrogen-bond to cellulose and to complex with iodine. It was almost totally resistant to digestion by cellulase [endo-(1-->4)-beta-glucanase] and did not serve as a donor substrate for xyloglucan endotransglucosylase (XET) activity. Like several other anionic polysaccharides, it promoted XET activity when unmodified (non-ionic) xyloglucan was used as donor substrate. Anionic xyloglucan may mimic polyanions whose presence in the plant cell wall promotes the action of endogenous XTH proteins. NaOCl with TEMPO oxidised the heptasaccharide, XXXG, to form XXX-glucarate, which did serve as an acceptor substrate although at a rate approximately fourfold less than XXXG itself. Anionic derivatives of xyloglucan, acting as acceptor but not donor substrates, may be valuable tools for exploring the biological roles of XTHs in the integration versus the re-structuring of xyloglucan in the plant cell wall.

Chemical structure of kenaf xylan

Methylation and partial acid hydrolysis of xylans from the bast and core of kenaf ( Hibiscus cannabinus) showed that the main chain of these xylans consists of (1 → 4)-linked β- d-xylopyranosyl (Xyl p) residues, some of which carry a α-1,2-linked 4- O-methyl-glucopyranosyluronic acid (Me-GlcA p) and glucopyranosyluronic acid (GlcA p) residues as side chains. Partial hydrolysis of kenaf xylans afforded two series of aldouronic acids from aldobio- to aldotetraouronic acids. The acids of the first series composed of 4- O-Me- d-GlcA p and d-Xyl p residues: 4- O-Me-GlcA-Xyl 3, 4- O-Me-GlcA-Xyl 2 and 4- O-Me-GlcA-Xyl. The second series composed of d-GlcA p and d-Xyl p: GlcA-Xyl 3, GlcA-Xyl 2 and GlcA-Xyl. In addition to these acids, another aldobiouronic acid, 4- O-(α- d-GalA p)- d-Xyl was found to be present in the partial hydrolysate. The molar ratio of GalA, GlcA, 4- O-Me-GlcA, and Xyl residues was calculated to be 1.0:2.0:9.4:119 for the bast xylan and 1.0:1.3:7.9:99.4 for the core xylan.

Culture conditions for the production of an acidic exopolysaccharide by the nitrogen-fixing bacterium Burkholderia tropica

The endophytic diazotrophic bacterium Burkholderia tropica, strain Ppe8, produced copious amounts of exopolysaccharide (EPS) on batch growth in liquid synthetic media containing mannitol and glutamate as carbon and nitrogen sources. The effect of various aeration regimes and carbon source concentrations on EPS production was determined, as well as the effects of temperature and time of growth. The degree of aeration had a great influence on the yield of EPS, in contrast with the C:N ratio of the medium. Growth temperature also affected the EPS yield after the first 24 h of culture but seemed to be irrelevant after that. After isolation and purification, the EPS was analyzed by high-performance size exclusion chromatography and multiangle laser light scattering (HPSEC-MALLS), revealing a molecular mass of 300 kDa. The acid hydrolyzate of EPS was examined by HPLC and found to contain Glc, Rha, GlcA, and an aldobiouronic acid. The latter was found to have a GlcA and a Rha unit. Carboxy-reduced EPS contained Glc and Rha (3:2). The monosaccharide composition of the native acidic EPS was calculated as GlcA, Glc, and Rha in a molar ratio of 1:2:2.

Characterization of the Aureobasidium pullulans α-glucuronidase expressed in Saccharomyces cerevisiae

The α-glucuronidase gene ( aguA) of Aureobasidium pullulans NRRL Y-2311-1 was amplified by PCR and sequenced. Based on its deduced amino acid sequence, AguA was found to be a member of family 67 of the glycoside hydrolases. It shares greater than 60% identity and between 34% and 42% identity with fungal and with bacterial α-glucuronidases, respectively. The open reading frame lacks introns and encodes a polypeptide of 836 amino acids that contains a putative signal peptide of 15 amino acids resulting in a mature protein with a calculated molecular mass of 91.0 kDa. A construct of the aguA gene encoding an additional C-terminal hexahistidine tag was cloned on an episomal plasmid under control of the ADH2 promoter and terminator and expressed in Saccharomyces cerevisiae Y294. The heterologous α-glucuronidase was purified to homogeneity by Ni-chelation affinity chromatography, and displayed an electrophoretic mobility of 157 kDa on SDS–PAGE. Maximal activity was measured at 65 °C and at pH 5 and pH 6. The enzyme had K m values in the millimolar range for the series of substrates from aldobiouronic acid to aldopentaouronic acid, but was unable to hydrolyze an internally substituted aldopentaouronic acid.

Alkaline degradation of model compounds related to beech xylan

Abstract To address the chemical behavior of beech xylan (O-acetyl-4-O-methyl-glucuronoxylan) under alkaline conditions, three model compounds, 2-O-methylxylobiose (1), aldobiouronic acid (4-O-methyl-α-D-glucopyranosyluronic acid-(1→2)-xylose, 2), and aldotriouronic acid (4-Omethyl-α-D-glucopyranosyluronic acid-(1→2)-β-D-xylopyranosyl-(1→4)-D-xylose, 3), were subjected to strong alkaline conditions equal to those used for the industrial production of viscose (18% NaOH, 43°C). Kinetics of the degradation of the model compounds were monitored by capillary electrophoresis in combination with pre-column derivatization. It was demonstrated that substitution at O-2 of the reducing xylose unit strongly retarded the alkaline degradation reactions (1 and 2). By isotopic labeling experiments and isolation of degradation products it was shown that under the pertinent conditions deprotonation at C-2 occurs, followed by epimerization to the respective lyxo derivative. Aldotriouronic acid 3 was degraded to 2 as an intermediate according to classical peeling pathways. Genuine degradation reactions and epimerization processes were distinguished.

An alkali-soluble heteroxylan from seeds of Phoenix dactylifera L.

Alkali-soluble polysaccharides, isolated from the seeds of dates, have been investigated using methylation and partial hydrolysis studies. The polysaccharides are shown to contain d-xylose and 4- O-methyl- d-glucuronic acid in a molar ratio of 5:1. An aldobiouronic acid from hemicellulose was characterized, and investigation revealed that the hemicellulose consists of a polymer of (1→4)-linked d-xylopranosyl residues having branches of d-xylopyranosyl and 4- O-methyl-α- d-glucopyranosyluronic acid.

The α-Glucuronidase, GlcA67A, of Cellvibrio japonicus Utilizes the Carboxylate and Methyl Groups of Aldobiouronic Acid as Important Substrate Recognition Determinants

alpha-Glucuronidases are key components of the ensemble of enzymes that degrade the plant cell wall. They hydrolyze the alpha1,2-glycosidic bond between 4-O-methyl-d-glucuronic acid (4-O-MeGlcA) and the xylan or xylooligosaccharide backbone. Here we report the crystal structure of an inactive mutant (E292A) of the alpha-glucuronidase, GlcA67A, from Cellvibrio japonicus in complex with its substrate. The data show that the 4-O-methyl group of the substrate is accommodated within a hydrophobic sheath flanked by Val-210 and Trp-160, whereas the carboxylate moiety is located within a positively charged region of the substrate-binding pocket. The carboxylate side chains of Glu-393 and Asp-365, on the "beta-face" of 4-O-MeGlcA, form hydrogen bonds with a water molecule that is perfectly positioned to mount a nucleophilic attack at the anomeric carbon of the target glycosidic bond, providing further support for the view that, singly or together, these amino acids function as the catalytic base. The capacity of reaction products and product analogues to inhibit GlcA67A shows that the 4-O-methyl group, the carboxylate, and the xylose sugar of aldobiouronic acid all play an important role in substrate binding. Site-directed mutagenesis informed by the crystal structure of enzyme-ligand complexes was used to probe the importance of highly conserved residues at the active site of GlcA67A. The biochemical properties of K288A, R325A, and K360A show that a constellation of three basic amino acids (Lys-288, Arg-325, and Lys-360) plays a critical role in binding the carboxylate moiety of 4-O-MeGlcA. Disruption of the apolar nature of the pocket created by Val-210 (V210N and V210S) has a detrimental effect on substrate binding, although the reduction in affinity is not reflected by an inability to accommodate the 4-O-methyl group. Replacing the two tryptophan residues that stack against the sugar rings of the substrate with alanine (W160A and W543A) greatly reduced activity.

Structural Analysis of the Extracellular Polysaccharide Produced bySphaerotilus natans

An extracellular polysaccharide (EPS) was recovered and purified from the culture fluid of a sheathed bacterium, Sphaerotilus natans. Glucose, rhamnose, and aldobiouronic acid were detected in the acid hydrolysate of EPS by thin-layer chromatography (TLC). The aldobiouronic acid was found to be composed of glucuronic acid and rhamnose by TLC and gas-liquid chromatography analyses of the corresponding neutral disaccharide. The structure of EPS was identified by methylation linkage analysis and nuclear magnetic resonance. Additionally, partial acid hydrolysates of EPS were prepared and put through fast atom bombardment-mass spectrometry to determine the sugar sequence of EPS. The resulting data showed that EPS produced by S. natans is a new gellan-like polysaccharide constructed from a tetrasaccharide repeating unit, as shown below. -->4)-alpha-D-Glcp-(1-->2)-beta-D-GlcA p-(1-->2)-alpha-L-Rha p-(1-->3)-beta-L-Rha p-(1-->.

Relevant structural features of the polysaccharide from Pithecellobium mangense gum exudate

Pithecellobium mangense (Leguminosae), disseminated in north-east of Venezuela, produces a clear gum very soluble in water. The polysaccharide, isolated from this gum, contains galactose, arabinose and glucuronic acid and its 4- o-methyl derivative. Smith degradation analysis indicated that the backbone structure is that of a β-(1→3) galactan. Methylation analysis revealed the presence of 3,6-di- o-substituted galactosyl units, indicating a branched structure. Arabinosyl side chains, up to four units long, were present as α- l-arabinofuranosyl (terminal and 3- o-linked) and β- l-arabinopyranosyl (3- o-linked) residues. Two aldobiouronic acids, 6- o-β- d-glucopyranosyluronic acid- d-galactose and 4- o-(4- o-methyl-α- d-glucuronopyranosyl)- d-galactose, were isolated and characterized.

Improved synthesis of an aldobiouronic acid related to hardwood xylans, and preparation of a derivative thereof suitable for linking to proteins

Treatment of 1,3,4-tri- O-acetyl- α- d-xylopyranose with methyl 2,3-di- O-benzyl-1-chloro-1-deoxy-4- O-methyl- α, β- d-glucopyranuronate in the presence of silver trifluoromethanesulfonate was highly stereoselective to give the α-linked aldobiouronic acid derivative ( 4) in 86% yield, after hydrogenolysis of the crude product of the coupling and chromatography. Compound 4 was acetylated and the fully protected substance was converted to the corresponding glycosyl chloride. Reaction of the latter with p-nitrophenol under phase-transfer catalysis afforded, after deacetylation, p-nitrophenyl 2- O-(methyl 4- O-methyl- α- d-glucopyranosyluronate)- β- d-xylopyranoside.

NMR analysis of the chemical structure of ulvan and of ulvan-boron complex formation

The NMR spectroscopic analysis of autohydrolysate fragments obtained from the gel-forming water-soluble cell-wall sulphated polysaccharides from the green seaweed Ulva sp. allowed the identification of two major repeating units in ulvan. These were the aldobiouronic acids → 4)-β-DGl cpA-(1 → 4)-α-L-Rha p 3-sulphate and → 4)-α-L-Ido pA-(l → 4)-α-L-Rha p 3-sulphate that were given the name ulvanobiouronic acid 3-sulphate A and B, respectively. These structures failed to bind boric acid since no ulvan-boron complex was observed by 11B or 13C NMR spectroscopy at the optimum pH of ulvan gelation (7.5). Boron complexes were only observed at pH ≥ 9 with ulvanobiouronic acid A. These results indicate that the ring hydroxyl groups of the major repeating structures which contain the rarely encountered iduronic acid in plant polysaccharides are probably not involved in the boric acid fixation step during the gelation of ulvan.

Sugar determination in ulvans by a chemical-enzymatic method coupled to high performance anion exchange chromatography

The sugar determination of ulvans, the water-soluble polysaccharides from Ulva sp. and Enteromorpha sp., was optimized by combining partial acid prehydrolysis (2 mol L-1 trifluoroacetic acid, 120°C) with enzymic hydrolysis (with β-D-glucuronidase). The different constitutive sugars (rhamnose, galactose, glucose, xylose, glucuronic acid), released after hydrolysis, were separated by high performance anion-exchange chromatography and determined by pulsed amperometric detection. The ulvanobiouronic acid, β-D-GlcA-(1,4)-L-Rha, which is the main constituent of ulvans was always present after 3 h of trifluoroacetic acid hydrolysis (approx. 2% D.M.) when acid hydrolysis was performed alone but the xylose amount fell to 75% of its maximum value at this time. The optimal duration of 2 mol L−1 trifluoroacetic acid hydrolysis of ulvans (i.e. without any degradation of xylose, rhamnose and glucuronic acid) was 45 min. Additionnal treatment of the partial acid hydrolysate by purified β-D-glucuronidase allowed the hydrolysis of the residual ulvanobiouronic acid in rhamnose and glucuronic acid. High performance anion exchange chromatography coupled to this chemical-enzymic hydrolysis revealed to be a high resolution chromatographic technique for monitoring the hydrolysis of the aldobiouronic acid by β-D-glucuronidase. This method allowed the simultaneous quantitative determination of neutral and acidic sugars and revealed the presence of iduronic acid inulvans.

Alpha-(4-O-methyl)-D-glucuronidase activity produced by the rumen anaerobic fungus Piromonas communis: a study of selected properties.

The rumen anaerobic fungus Piromonas communis, unlike the rumen anaerobic fungi Neocallimastix frontalis and Neocallimastix patriciarum, produced extracellular alpha-(4-O-methyl)-D-glucuronidase when grown in cultures containing filter-paper, barley straw, birchwood xylan or birchwood sawdust as carbon source. The highest concentration of enzyme was produced in cultures containing birchwood sawdust. The aldobiouronic acid O-alpha-(4-O-methyl-D-glucopyranosyluronic acid)-(1-->2)-D-xylopyranose (MeGlcAXyl) was the best substrate of those tested: the aldotriouronic acid O-alpha-(4-O-methyl-D-glucopyranosyluronic acid (1-->2)-O-beta-D-xylopyranosyl-(1-->4)-D-xylopyranose (MeGlcAXyl2) and the aldotetraouronic acid O-alpha-(4-O-methyl-D-glucopyranosyluronic acid)-(1-->2)-O-beta-D- xylopyranosyl-(1-->4)-O-beta-D-xylopyranosyl-(1-->4)-D-xylopyranose (MeGlcAXyl3) were also attacked but the rate fell as the degree of polymerisation increased. When the same substituted xylo-oligosaccharides were reduced to the corresponding alditols the enzyme activity disappeared. Similarly, p-nitrophenyl-alpha-D-glucuronide was not a substrate. Remarkably, the relative rates of attack shown by the alpha-(4-O-methyl)-D-glucuronidase on the aldouronic acids and on xylans extracted from birchwood, oat spelts and oat straw differed according to the carbon source used to produce the enzyme. The alpha-(4-O-methyl)-D-glucuronidase had a pH optimum of 5.5 and a temperature optimum of 50 degrees C. On gel filtration the enzyme was shown to be associated with proteins covering the range 100-300 kDa, but a major peak of activity in the column effluent appeared to have a molecular mass of 103 kDa.

Structure of chia seed polysaccharide exudate

Chia seed gum varies in molecular weight from 0·8 to 2·0 × 10 6 daltons, as determined by gel filtration. β- d-Xylose, α- d-glucose, and 4-O- methyl-α- d-glucuronic acid were obtained on hydrolysis in the respective ratios of 2:1:1. An aldobiouronic acid, 2-O-(4-O- methyl-α- d-glucopyranosiduronic acid)- d-xylose , was obtained by partial hydrolysis. A tentative structural unit proposed for the polysaccharide is a tetrasaccharide with 4-O- methyl-α- d-glucoronopyranosyl residues occurring as branches at O-2 of some β- d-xylopyranosyl residues in the main chain consisting of (1→4)-β- d-xylopyranosyl-(1→4)-α- d-glucopyranosyl-(1→4)-β- d-xylopyranosyl units. A linear tetrasaccharide was characterized that incorporates these structural features.

Stereoselective synthesis of regioisomers of aldobiouronic acid.

alpha-Glucuronidase is a very important enzyme for the complete hydrolysis of plant hemicellulose, but the substrate specificity of the enzyme has not previously been reported. In this connection, the three regioisomers of O-(alpha-D-glucopyranosyluronic acid)-D-xylose (aldobiouronic acid), 2-O-(alpha-D-glucopyranosyluronic acid)-D-xylose (13), 3-O-(alpha-D-glucopyranosyluronic acid)-D-xylose (14), and 4-O-(alpha-D-glucopyranosyluronic acid)-D-xylose (15), were stereoselectively synthesized to clarify the substrate specificity.