Xyloglucan Oligomers Research Articles

Bioinformatics-based identification of GH12 endoxyloglucanases in citrus-pathogenic Penicillium spp

Millions of tons of citrus peel waste are produced every year as a byproduct of the juice industry. Citrus peel is rich in pectin and xyloglucan, but while the pectin is extracted for use in the food industry, the xyloglucan is currently not valorized. To target hydrolytic degradation of citrus peel xyloglucan into oligosaccharides, we have used bioinformatics to identify three glycoside hydrolase 12 (GH12) endoxyloglucanases (EC 3.2.1.151) from the citrus fruit pathogens Penicillium italicum GL-Gan1 and Penicillium digitatum Pd1 and characterized them on xyloglucan obtained by alkaline extraction from citrus peel. The enzymes displayed pH-temperature optima of pH 4.6–5.3 and 35–37°C. PdGH12 from P. digitatum and PiGH12A from P. italicum share 84% sequence identity and displayed similar kinetics, although kcat was highest for PdGH12. In contrast, PiGH12B from P. italicum, which has the otherwise conserved Trp in subsite −4 replaced with a Tyr, displayed a 3 times higher KM and a 4 times lower kcat/KM than PiGH12A, but was the most thermostable enzyme of the three Penicillium-derived endoxyloglucanases. The benchmark enzyme AnGH12 from Aspergillus nidulans was more thermally stable and had a higher pH-temperature optimum than the enzymes from Penicillum spp. The difference in structure of the xyloglucan oligosaccharides extracted from citrus peel xyloglucan and tamarind xyloglucan by the new endoxyloglucanases was determined by LC-MS. The inclusion of citrus peel xyloglucan demonstrated that the endoxyloglucanases liberated fucosylated xyloglucan oligomers, implying that these enzymes have the potential to upgrade citrus peel residues to produce oligomers useful as intermediates or bioactive compounds.

Entropy drives the adsorption of xyloglucan to cellulose surfaces – A molecular dynamics study

The adsorption of nonionic polymers to cellulose is of large importance both in the plant cell wall during synthesis and for the development of sustainable materials from wood. Here, the thermodynamics of adsorption of the polysaccharide xyloglucan (XG) to both native and chemically modified cellulose with carboxyl groups was investigated using molecular dynamics simulations. The free energy of adsorption was calculated as the potential of mean force between an XG oligomer and model cellulose surfaces in a range of temperatures from 298 K to 360 K. It was found that the adsorption near room temperature is an endothermic process dominated by the entropy of released interfacial water molecules. This was corroborated by quantitative assessment of the absolute entropy per water molecule both at the interface and in the bulk. In the case of native cellulose, the adsorption became exothermic at higher temperatures, while the relatively strong interactions between water and the charged groups of the oxidized cellulose impede such a transition. The results also indicate that the extraction of strongly associated hemicelluloses would be facilitated by low temperature.

Molecular insights of a xyloglucan endo-transglycosylase/hydrolase of radiata pine (PrXTH1) expressed in response to inclination: Kinetics and computational study

Xyloglucan endotransglycosylase/hydrolases (XTH) may have endotransglycosylase (XET) and/or hydrolase (XEH) activities. Previous studies confirmed XET activity for PrXTH1 protein from radiata pine. XTHs could interact with many hemicellulose substrates, but the favorite substrate of PrXTH1 is still unknown. The prediction of union type and energy stability of the complexes formed between PrXTH1 and different substrates (XXXGXXXG, XXFGXXFG, XLFGXLFG and cellulose) were determined using bioinformatics tools. Molecular Docking, Molecular Dynamics, MM-GBSA and Electrostatic Potential Calculations were employed to predict the binding modes, free energies of interaction and the distribution of electrostatic charge. The results suggest that the enzyme formed more stable complexes with hemicellulose substrates than cellulose, and the best ligand was the xyloglucan XLFGXLFG (free energy of −58.83 ± 0.8 kcal mol−1). During molecular dynamics trajectories, hemicellulose fibers showed greater stability than cellulose. Aditionally, the kinetic properties of PrXTH1 enzyme were determined. The recombinant protein was active and showed an optimal pH 5.0 and optimal temperature of 37 °C. A Km value of 20.9 mM was determined for xyloglucan oligomer. PrXTH1 is able to interact with different xyloglycans structures but no activity was observed for cellulose as substrate, remodeling cell wall structure in response to inclination.

Xyloglucan O-acetyltransferases from Arabidopsis thaliana and Populus trichocarpa catalyze acetylation of fucosylated galactose residues on xyloglucan side chains.

AXY4/XGOAT1, AXY4L/XGOAT2 and PtrXGOATs are O-acetyltransferases acetylating fucosylated galactose residues on xyloglucan and AXY9 does not directly catalyze O-acetylation of xyloglucan but exhibits weak acetylesterase activity. Xyloglucan is a major hemicellulose that cross-links cellulose in the primary walls of dicot plants and the galactose (Gal) residues on its side chains can be mono- and di-O-acetylated. In Arabidopsis thaliana, mutations of three AXY (altered xyloglucan) genes, AXY4, AXY4L and AXY9, have previously been shown to cause a reduction in xyloglucan acetylation, but their biochemical functions remain to be investigated. In this report, we demonstrated that recombinant proteins of AXY4/XGOAT1 (xyloglucan O-acetyltransferase1), AXY4L/XGOAT2 and their close homologs from Populus trichocarpa, PtrXGOATs, displayed O-acetyltransferase activities transferring acetyl groups from acetyl CoA onto xyloglucan oligomers. Structural analysis of XGOAT-catalyzed reaction products revealed that XGOATs mediated predominantly 6-O-monoacetylation and a much lesser degree of 3-O and 4-O-monoacetylation and 4,6-di-O-acetylation of Gal residues on xyloglucan side chains. XGOATs appeared to preferentially acetylate fucosylated Gal residues with little activity toward non-fucosylated Gal residues. Mutations of the conserved amino acid residues in the GDS and DXXH motifs in AXY4/XGOAT1 resulted in a drastic reduction in its ability to transfer acetyl groups onto xyloglucan oligomers. In addition, although recombinant AXY9 was unable to transfer acetyl groups from acetyl CoA onto xyloglucan oligomers, it was catalytically active as demonstrated by its weak acetylesterase activity that was also exhibited by AXY4/XGOAT1 and AXY4L/XGOAT2. Furthermore, we showed that the AXY8 fucosidase was able to hydrolyze fucosyl residues from both non-acetylated and acetylated xyloglucan oligomers. These findings provide biochemical evidence that AXY4/XGOAT1, AXY4L/XGOAT2 and PtrXGOATs are xyloglucan O-acetyltransferases catalyzing acetyl transfer onto fucosylated Gal residues on xyloglucan side chains and the defucosylation of these acetylated side chains by apoplastic AXY8 generates side chains with acetylated, non-fucosylated Gal residues.

Enzymatically Activated Glyco-Prodrugs of Doxorubicin Synthesized by a Catalysis-Free Diels-Alder Reaction.

The severe side effects associated with the use of anthracycline anticancer agents continues to limit their use. Herein we describe the synthesis and preliminary biological evaluation of three enzymatically activatable doxorubicin-oligosaccharide prodrugs. The synthetic protocol allows late stage variation of the carbohydrate and is compatible with the use of disaccharides such as lactose as well as more complex oligosaccharides such as xyloglucan oligomers. The enzymatic release of doxorubicin from the prodrugs by both protease (plasmin) and human carboxylesterases (hCE1 and 2) was demonstrated in vitro and the cytotoxic effect of the prodrugs was assayed on MCF-7 breast cancer cells.

Glycosylation is important for FcXTH1 activity as judged by its structural and biochemical characterization

Xyloglucan endotransglycosylase/hydrolases (XTH) may have endotransglycosylase (XET) and/or hydrolase (XEH) activities. Previous studies suggest that XTHs might play a key role in ripening of Fragaria chiloensis fruit as FcXTH1 transcripts increase as fruit softens. FcXTH1 protein sequence contains a conserved N-glycosylation site adjacent to catalytic residues. The FcXTH1 structure was built through comparative modeling methodology, the structure displays a β-jellyroll–type folding with a curvature generated by eight antiparallel β-sheets that holds the catalytic motif that is oriented towards the central cavity of the protein. Through Molecular Dynamic Simulations (MDS) analyses the protein-ligand interactions of FcXTH1 were explored, finding a better interaction with xyloglucans than cellulose. Nevertheless, the stability of the protein-ligand complex depends on the glycosylation state of FcXTH1: better energy interactions were determined for the glycosylated protein. As a complement, the molecular cloning and heterologous expression of FcXTH1 in Pichia pastoris was performed, and the recombinant protein was active and displayed strict XET activity. A KM value of 17.0 μM was determined for xyloglucan oligomer. The deglycosylation of FcXTH1 by PNGase-F treatment affects its biochemical properties (increase KM and reduce kcat/KM ratio) and reduces its stability. As a conclusion, glycosylation of FcXTH1 is important for its biological function.

Swelling and dimensional stability of xyloglucan/montmorillonite nanocomposites in moist conditions from molecular dynamics simulations

Nacre-mimetic biocomposites made from the combination of montmorillonite clay and the hemicellulose xyloglucan give materials that retain much of their material properties even at high relative humidity. Here, a model composite system consisting of two clay platelets intercalated by xyloglucan oligomers was studied at different levels of hydration using molecular dynamics simulations, and compared to the pure clay. It was found that xyloglucan inhibits swelling of the clay at low water contents by promoting the formation of nano-sized voids that fill with water without affecting the material’s dimensions. At higher water contents the XG itself swells, but at the same time maintaining contact with both platelets across the gallery, thereby acting as a physical cross-linker in a manner similar to the role of XG in the plant cell wall.

Carbohydrate binding module recognition of xyloglucan defined by polar contacts with branching xyloses and CH-Π interactions.

Engineering of novel carbohydrate-binding proteins that can be utilized in various biochemical and biotechnical applications would benefit from a deeper understanding of the biochemical interactions that determine protein-carbohydrate specificity. In an effort to understand further the basis for specificity we present the crystal structure of the multi-specific carbohydrate-binding module (CBM) X-2 L110F bound to a branched oligomer of xyloglucan (XXXG). X-2 L110F is an engineered CBM that can recognize xyloglucan, xylans and β-glucans. The structural observations of the present study compared with previously reported structures of X-2 L110F in complex with linear oligomers, show that the π-surface of a phenylalanine, F110, allows for interactions with hydrogen atoms on both linear (xylopentaose and cellopentaose) and branched ligands (XXXG). Furthermore, X-2 L110F is shown to have a relatively flexible binding cleft, as illustrated in binding to XXXG. This branched ligand requires a set of reorientations of protein side chains Q72, N31, and R142, although these residues have previously been determined as important for binding to xylose oligomers by mediating polar contacts. The loss of these polar contacts is compensated for in binding to XXXG by polar interactions mediated by other protein residues, T74, R115, and Y149, which interact mainly with the branching xyloses of the xyloglucan oligomer. Taken together, the present study illustrates in structural detail how CH-π interactions can influence binding specificity and that flexibility is a key feature for the multi-specificity displayed by X-2 L110F, allowing for the accommodation of branched ligands.

Analysis of xyloglucans by ambient chloride attachment ionization tandem mass spectrometry

Xyloglucan oligomers obtained upon enzyme digestion from Hymenaea courbaril, Arabidopsis Columbia-0 and mur3 were ionized and analyzed by using chloride anion attachment electrospray ionization (ESI) and tandem mass spectrometry. MW determination and structural elucidation of several xyloglucan oligomers was performed directly from the mixture solutions without sample pretreatment or derivatization. Sodium cation attachment was used to determine the number of xyloglucans present in the mixtures and their MWs. However, tandem mass spectrometry results showed that structure elucidation based on the sodium adducts is ambiguous. Chloride anion also forms stable adducts with these xyloglucans upon ESI. These adducts can be readily identified due to the chlorine isotope pattern. The mass spectral profile of xyloglucans obtained for the mixtures matches the HPAEC results, thus validating this methodology for the determination of the xyloglucan composition and the MW of each xyloglucan. Upon collisional activation in MS2 experiments, the chloride anion adducts readily lose HCl, which helps verify the molecular weight of each xyloglucan. Isolating the resulting anion (deprotonated oligomer) and subjecting it to further collision-activated dissociation experiments (MSn; n=3–4) yields useful structural information that allows the differentiation between isomeric anions and hence determination of the sequence of the xyloglucan oligomers. The deprotonated oligomers fragment by a stepwise loss of sugar units from the reducing end.

Oligosaccharide mass profiling of nutritionally important Salicornia brachiata, an extreme halophyte

Salicornia brachiata is an extreme halophyte, growing opulently in salt marshes and considered as potential alternative crop for seawater agriculture. Salicornia seeds are rich in protein and tender-shoots are eaten as salad greens. Total cell wall carbohydrate was isolated from seedlings, digested with β-glucanase enzyme cocktail and oligosaccharide mass profiling (OLIMP) was performed by using MALDI TOF-TOF mass spectroscopy. Salicornia OLIMP is represented by characteristic mass peaks m/z 477.3297–2094.4363. MS spectra exhibit xyloglucan oligosaccharide building blocks, dominated by XXXG (1084.9927m/z). Characteristic mass peaks (m/z) of xyloglucan oligomers (XXG, XXFG, XLXG and XLFG) were also detected, which showed resemblance to the mass profile of highly nutritious plant soybean. The present study is the first report on OLIMP for any edible halophyte, so far. OLIMP supports use of Salicornia as a potential source of dietary supplementation. Further linkage-analysis is required to get the structural information of oligosaccharides.

Cloning, expression, and characterization of an oligoxyloglucan reducing end-specific xyloglucanobiohydrolase from Aspergillus nidulans

An oligoxyloglucan reducing end-specific xyloglucanobiohydrolase from the filamentous fungus Aspergillus nidulans was cloned and expressed in Pichia pastoris as a secreted histidine-tagged protein and purified by affinity chromatography. The enzyme acts on xyloglucan oligomers and releases the first two glycosyl residue segments from the reducing end, provided that neither the first glucose nor the xylose attached to the third glucose residue from the reducing end is not further substituted. The enzyme has a specific activity of 7 U/mg at the pH optimum of 3 and at the temperature optimum of 42 °C.

Structural investigation of hemicellulosic polysaccharides from Argania spinosa: characterisation of a novel xyloglucan motif

Hemicellulose polymers were isolated from Argania spinosa leaf cell walls by sequential extractions with alkali. The structure of the two main polymers, xylan and xyloglucan, was investigated by enzyme degradation with specific endoglycosidases followed by analysis of the resulting fragments by high performance anion exchange chromatography (HPAEC) and matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS). The results show that A. spinosa xylan is composed of a β-(1 → 4)-linked- d-xylopyranose backbone substituted with 4- O-methyl- d-glucuronic acid residues. Xyloglucan oligosaccharide subunits were generated by treatment with an endo-(1 → 4)-β- d-glucanase of the xyloglucan-rich hemicellulosic fractions. MALDI-TOF mass spectra and HPAE-PAD chromatography of the pool of endoglucanase-generated xyloglucan oligomers indicated that A. spinosa cell wall contains a XXXG-type xyloglucan. In addition to XXXG, XXFG, XLXG/XXLG, XLFG fragments previously characterised in various plants, a second group of XXXG-type fragments was detected. The primary structure of the major subunit was determined by a combination of sugar analysis, methylation analysis, post-source decay (PSD) fragment analysis of MALDI-TOF MS and 1H NMR spectroscopy. This fragment, termed XUFG, contains a novel β- d-Xyl p-(1 → 2)-α- d-Xyl p side chain linked to C-6 of the second glucose unit from the nonreducing end of the cellotetraose sequence.

Analysis of retted and non retted flax fibres by chemical and enzymatic means

Flax fibres ( Linum usitatissimum L.) were subjected to chemical and enzymatic analysis in order to determine the compositional changes brought about by the retting process and also to determine the accessibility of the fibre polymers to enzymatic treatment. Chemical analysis involved subjecting both retted and non retted fibres to a series of sequential chemical extractions with 1% ammonium oxalate, 0.05 M KOH, 1 M KOH and 4 M KOH. Retting was shown to cause minimal weight loss from the fibres but caused significant changes to the pectic polymers present. Retted fibres were shown to have significantly lower amounts of rhamnogalacturonan as well as arabinan and xylan. In addition the average molecular mass of the pectic extracts was considerably lowered. Enzyme treatment of the 1 M KOH extracts with two different enzymes demonstrated that the non retted extract contained a relatively high molecular weight xylan not found in the retted extract. Treatment of the 1 M KOH extracts and the fibres with Endoglucanase V from Trichoderma viride demonstrated that while this enzyme solubilised cellulose as well as xylan and xyloglucan oligomers from the extract, it had limited access to these polymers on the fibre. MALDI-TOF MS analysis of the material solubilised from the extract suggested that the xylan was randomly substituted with 4-O-methyl glucuronic acid moieties. The xyloglucan was shown to be of the XXXG type and was substituted with galactose and fucose units. The enzyme treatments of the fibres demonstrated that the xylan and xyloglucan polymers in the fibres were not accessible to the enzyme but that material which was entrapped by the cellulose could be released by the hydrolysis of this cellulose.

A conformational study of the xyloglucan oligomer, XXXG, by NMR spectroscopy and molecular modeling

A structural study of the XXXG xyloglucan heptasaccharide (X = α-D-Xylp(1 → 6)-β-D-Glcp and G = β-D-Glcp) isolated from apple fruit has been undertaken with nmr and molecular mechanics methods. Quantitative 400 MHz nmr data including nuclear Overhauser effect spectroscopy (NOESY) volumes were recorded at both 6 and 20°C. In spite of severe overlapping of resonances, it was possible to estimate summed NOEs for the majority of the anomeric and glucosyl methylene protons. An ensemble-average population of preferred geometries has been established with the CICADA conformational searching algorithm associated with the MM3 force field. Comparison of the theoretical data obtained by back-calculation of the NOESY volumes from the ensemble-average distance matrix program and motional models based on the Stokes–Einstein–Debye relation satisfactorily reproduce the experimental data. Conformational averaging about the mainchain glycosidic linkages includes both the syn and anti conformers and a minor gauche–gauche population is highly probable. The theoretical data overestimate the syn preference of the Glcc → Glcb linkage as well as the Glcc GT rotamer population. Finally, both the motional models and the conformational search indicate a fairly rigid backbone and greater flexiblity for the xylose side chains. © 2000 John Wiley & Sons, Inc. Biopoly 54: 11–26, 2000

Xyloglucan from soybean ( Glycine max) meal is composed of XXXG-type building units

Soybean cell wall material was depectinated by extraction with a hot chelating agent and a cold dilute alkali. The hemicelluloses were solubilised from the residue with 1 and 4 M KOH solutions, resulting in 1 M Alkali Soluble Solids (1 MASS) and 4 M Alkali Soluble Solids (4 MASS) fractions. The polysaccharides extracted with 1 M KOH were fractionated by ion-exchange chromatography, yielding a neutral and a pectic population. The sugar composition of the neutral population indicated the presence of xyloglucans and possibly xylans. Enzymatic degradation with endo-xylanases and endo-glucanases showed the presence of xyloglucans only. Analysis of the digest formed after incubation of the neutral population with endo-glucanase V using both HPAEC and MALDI-TOF MS showed the formation of the characteristic poly-XXXG xyloglucan oligomers (XXG, XXXG, XXFG, XLXG, and XLFG).

Conformation of Tamarind Seed Xyloglucan Oligomers.

Tamarind seed polysaccharide (TSP) xyloglucan consists of a (1→4)-β-D-glucan spine (cellulose spine) with (1→6)-α-branched xylose, which is partially substituted by (1→2) -β-galacto-xylose. Thus TSP xyloglucan yields three types of monomer units (heptasaccharide DP7, octasaccharide DP8 and nonasaccharide DP9 where DPdenotes the number of residues), which differs in the number of galactose side chains. Three types of monomers were prepared and their conformation in water was elucidated in detail by means of small-angle X-ray scattering and molecular dynamics. Dimers, trimers and tetramers (constituted of a mixture of arbitrary combinations of three types of monomers) were also prepared and their conformation was analyzed from the results of small-angle X-ray scattering. The results confirm the thin ribbon-like structure of xyloglucan oligomers. The conformation of xylose and galacto-xylose branches was also discussed.

Visualization of EXGT-Mediated Molecular Grafting Activity by Means of a Fluorescent-Labeled Xyloglucan Oligomer

A fluoresceinyl xyloglucan heptasaccharide was synthesized and used as a probe to visualize the activity of the cell wall to incorporate xyloglucan fragments into the cellulose/xyloglucan framework in suspension-cultured tobacco cells. The activity was significantly reduced in transformant cells in which the expression of endoxyloglucan transferase (EXGT) was severely suppressed by over-expression of the antisense EXGT-N1 mRNA. These results indicate the involvement of EXGT in the construction of the cellulose/xyloglucan framework.

High-performance reversed-phase chromatographic mapping of 2-pyridylamino derivatives of xyloglucan oligosaccharides

Xyloglucan oligosaccharides from cotton cell walls and tamarind seeds were derivatized with 2-aminopyridine and subsequently separated by reversed-phase chromatography (r.p.c.) using an octadecylsilyl silica stationary phase and aqueous-organic eluents with 0.01% (v/v) trifluoroacetic acid. The chromatographic behavior of the 2-pyridylamino derivatives of xyloglucan oligosaccharides was examined under a wide range of elution conditions, including gradient steepness and shape initial acetonitrile concentration in the eluent, and pore size of the r.p.c. packings. Relatively steep acetonitrile gradients resulted in poor resolution of the different xyloglucan fragments, which is believed to be the result of acetonitrile-induced conformational changes. Under these circumstances the elution order of the derivatized-xyloglucan oligosaccharides was such that the smaller fragments eluted from the column before the larger ones. R.p.c. packing with a 70-Å pore size necessitated relatively high acetonitrile concentration in the eluent when compared with 300-Å stationary phase. The r.p.c. mapping of 2-pyridylamino derivatives of xyloglucan oligosaccharides was best achieved when both a wide-pore octadecyl-silyl silica stationary phase and a shallow gradient with consecutive linear segments of increasing acetonitrile concentration in the eluent were employed. This combination yielded rapid r.p.c. maps of the xyloglucan fragments from different sources with high separation efficiencies and concomitantly high resolution. The effects of the nature of the sugar residues in the xyloglucan oligomers and their degree of branching on r.p.c. retention and selectivity are also highlighted.