Grass xylan structural variation suggests functional specialization and distinctive interaction with cellulose and lignin.

Xylan is the most abundant non-cellulosic polysaccharide in grass cell walls, and it has important structural roles. The name glucuronoarabinoxylan (GAX) is used to describe this variable hemicellulose. It has a linear backbone of ß-1,4-xylose (Xyl) residues that may be substituted with α-1,2-linked (4-O-methyl)-glucuronic acid (GlcA), α-1,3-linked arabinofuranose (Araf), and sometimes acetylation at the O-2 and/or O-3 positions. The role of these substitutions remains unclear, although there is increasing evidence that they affect the way xylan interacts with other cell wall components, particularly cellulose and lignin. Here, we used substitution-dependent endo-xylanase enzymes to investigate the variability of xylan substitution in grass culm cell walls. We show that there are at least three different types of xylan: (i) an arabinoxylan with evenly distributed Araf substitutions without GlcA (AXe); (ii) a glucuronoarabinoxylan with clustered GlcA modifications (GAXc); and (iii) a highly substituted glucuronoarabinoxylan (hsGAX). Immunolocalization of AXe and GAXc in Brachypodium distachyon culms revealed that these xylan types are not restricted to a few cell types but are instead widely detected in Brachypodium cell walls. We hypothesize that there are functionally specialized xylan types within the grass cell wall. The even substitutions of AXe may permit folding and binding on the surface of cellulose fibrils, whereas the more complex substitutions of the other xylans may support a role in the matrix and interaction with other cell wall components.

Altering the substitution and cross-linking of glucuronoarabinoxylans affects cell wall architecture in Brachypodium distachyon.

The Poaceae family of plants provides cereal crops that are critical for human and animal nutrition, and also, they are an important source of biomass. Interacting plant cell wall components give rise to recalcitrance to digestion; thus, understanding the wall molecular architecture is important to improve biomass properties. Xylan is the main hemicellulose in grass cell walls. Recently, we reported structural variation in grass xylans, suggesting functional specialisation and distinct interactions with cellulose and lignin. Here, we investigated the functions of these xylans by perturbing the biosynthesis of specific xylan types. We generated CRISPR/Cas9 knockout mutants in Brachypodium distachyon XAX1 and GUX2 genes involved in xylan substitution. Using carbohydrate gel electrophoresis, we identified biochemical changes in different xylan types. Saccharification, cryo-SEM, subcritical water extraction and ssNMR were used to study wall architecture. BdXAX1A and BdGUX2 enzymes modify different types of grass xylan. Brachypodium mutant walls are likely more porous, suggesting the xylan substitutions directed by both BdXAX1A and GUX2 enzymes influence xylan-xylan and/or xylan-lignin interactions. Since xylan substitutions influence wall architecture and digestibility, our findings open new avenues to improve cereals for food and to use grass biomass for feed and the production of bioenergy and biomaterials.

Subcritical water extraction of Equisetum arvense biomass withdraws cell wall fractions that trigger plant immune responses and disease resistance.

Plant cell walls are complex structures mainly made up of carbohydrate and phenolic polymers. In addition to their structural roles, cell walls function as external barriers against pathogens and are also reservoirs of glycan structures that can be perceived by plant receptors, activating Pattern-Triggered Immunity (PTI). Since these PTI-active glycans are usually released upon plant cell wall degradation, they are classified as Damage Associated Molecular Patterns (DAMPs). Identification of DAMPs imply their extraction from plant cell walls by using multistep methodologies and hazardous chemicals. Subcritical water extraction (SWE) has been shown to be an environmentally sustainable alternative and a simplified methodology for the generation of glycan-enriched fractions from different cell wall sources, since it only involves the use of water. Starting from Equisetum arvense cell walls, we have explored two different SWE sequential extractions (isothermal at 160 ºC and using a ramp of temperature from 100 to 160 ºC) to obtain glycans-enriched fractions, and we have compared them with those generated with a standard chemical-based wall extraction. We obtained SWE fractions enriched in pectins that triggered PTI hallmarks in Arabidopsis thaliana such as calcium influxes, reactive oxygen species production, phosphorylation of mitogen activated protein kinases and overexpression of immune-related genes. Notably, application of selected SWE fractions to pepper plants enhanced their disease resistance against the fungal pathogen Sclerotinia sclerotiorum. These data support the potential of SWE technology in extracting PTI-active fractions from plant cell wall biomass containing DAMPs and the use of SWE fractions in sustainable crop production.

Field testing of transgenic aspen from large greenhouse screening identifies unexpected winners.

Trees constitute promising renewable feedstocks for biorefinery using biochemical conversion, but their recalcitrance restricts their attractiveness for the industry. To obtain trees with reduced recalcitrance, large-scale genetic engineering experiments were performed in hybrid aspen blindly targeting genes expressed during wood formation and 32 lines representing seven constructs were selected for characterization in the field. Here we report phenotypes of five-year old trees considering 49 traits related to growth and wood properties. The best performing construct considering growth and glucose yield in saccharification with acid pretreatment had suppressed expression of the gene encoding an uncharacterized 2-oxoglutarate-dependent dioxygenase (2OGD). It showed minor changes in wood chemistry but increased nanoporosity and glucose conversion. Suppressed levels of SUCROSE SYNTHASE, (SuSy), CINNAMATE 4-HYDROXYLASE (C4H) and increased levels of GTPase activating protein for ADP-ribosylation factor ZAC led to significant growth reductions and anatomical abnormalities. However, C4H and SuSy constructs greatly improved glucose yields in saccharification without and with pretreatment, respectively. Traits associated with high glucose yields were different for saccharification with and without pretreatment. While carbohydrates, phenolics and tension wood contents positively impacted the yields without pretreatment and growth, lignin content and S/G ratio were negative factors, the yields with pretreatment positively correlated with S lignin and negatively with carbohydrate contents. The genotypes with high glucose yields had increased nanoporosity and mGlcA/Xyl ratio, and some had shorter polymers extractable with subcritical water compared to wild-type. The pilot-scale industrial-like pretreatment of best-performing 2OGD construct confirmed its superior sugar yields, supporting our strategy.

Glycogen synthase downregulation rescues the amylopectinosis of murine RBCK1 deficiency.

Longer glucan chains tend to precipitate. Glycogen, by far the largest mammalian glucan and the largest molecule in the cytosol with up to 55 000 glucoses, does not, due to a highly regularly branched spherical structure that allows it to be perfused with cytosol. Aberrant construction of glycogen leads it to precipitate, accumulate into polyglucosan bodies that resemble plant starch amylopectin and cause disease. This pathology, amylopectinosis, is caused by mutations in a series of single genes whose functions are under active study toward understanding the mechanisms of proper glycogen construction. Concurrently, we are characterizing the physicochemical particularities of glycogen and polyglucosans associated with each gene. These genes include GBE1, EPM2A and EPM2B, which respectively encode the glycogen branching enzyme, the glycogen phosphatase laforin and the laforin-interacting E3 ubiquitin ligase malin, for which an unequivocal function is not yet known. Mutations in GBE1 cause a motor neuron disease (adult polyglucosan body disease), and mutations in EPM2A or EPM2B a fatal progressive myoclonus epilepsy (Lafora disease). RBCK1 deficiency causes an amylopectinosis with fatal skeletal and cardiac myopathy (polyglucosan body myopathy 1, OMIM# 615895). RBCK1 is a component of the linear ubiquitin chain assembly complex, with unique functions including generating linear ubiquitin chains and ubiquitinating hydroxyl (versus canonical amine) residues, including of glycogen. In a mouse model we now show (i) that the amylopectinosis of RBCK1 deficiency, like in adult polyglucosan body disease and Lafora disease, affects the brain; (ii) that RBCK1 deficiency glycogen, like in adult polyglucosan body disease and Lafora disease, has overlong branches; (iii) that unlike adult polyglucosan body disease but like Lafora disease, RBCK1 deficiency glycogen is hyperphosphorylated; and finally (iv) that unlike laforin-deficient Lafora disease but like malin-deficient Lafora disease, RBCK1 deficiency's glycogen hyperphosphorylation is limited to precipitated polyglucosans. In summary, the fundamental glycogen pathology of RBCK1 deficiency recapitulates that of malin-deficient Lafora disease. Additionally, we uncover sex and genetic background effects in RBCK1 deficiency on organ- and brain-region specific amylopectinoses, and in the brain on consequent neuroinflammation and behavioural deficits. Finally, we exploit the portion of the basic glycogen pathology that is common to adult polyglucosan body disease, both forms of Lafora disease and RBCK1 deficiency, namely overlong branches, to show that a unified approach based on downregulating glycogen synthase, the enzyme that elongates glycogen branches, can rescue all four diseases.

Cell wall-derived mixed-linked ß-1,3/1,4-glucans trigger immune responses and disease resistance in plants.

Pattern-triggered immunity (PTI) is activated in plants upon recognition by pattern recognition receptors (PRRs) of damage- and microbe-associated molecular patterns (DAMPs and MAMPs) derived from plants or microorganisms, respectively. To understand better the plant mechanisms involved in the perception of carbohydrate-based structures recognized as DAMPs/MAMPs, we have studied the ability of mixed-linked ß-1,3/1,4-glucans (MLGs), present in some plant and microbial cell walls, to trigger immune responses and disease resistance in plants. A range of MLG structures were tested for their capacity to induce PTI hallmarks, such as cytoplasmic Ca2+ elevations, reactive oxygen species production, phosphorylation of mitogen-activated protein kinases and gene transcriptional reprogramming. These analyses revealed that MLG oligosaccharides are perceived by Arabidopsis thaliana and identified a trisaccharide, ß-d-cellobiosyl-(1,3)-ß-d-glucose (MLG43), as the smallest MLG structure triggering strong PTI responses. These MLG43-mediated PTI responses are partially dependent on LysM PRRs CERK1, LYK4 and LYK5, as they were weaker in cerk1 and lyk4 lyk5 mutants than in wild-type plants. Cross-elicitation experiments between MLG43 and the carbohydrate MAMP chitohexaose [ß-1,4-d-(GlcNAc)6 ], which is also perceived by these LysM PRRs, indicated that the mechanism of MLG43 recognition could differ from that of chitohexaose, which is fully impaired in cerk1 and lyk4 lyk5 plants. MLG43 treatment confers enhanced disease resistance in A. thaliana to the oomycete Hyaloperonospora arabidopsidis and in tomato and pepper to different bacterial and fungal pathogens. Our data support the classification of MLGs as a group of carbohydrate-based molecular patterns that are perceived by plants and trigger immune responses and disease resistance.

Comparison of Six Lytic Polysaccharide Monooxygenases from Thermothielavioides terrestris Shows That Functional Variation Underlies the Multiplicity of LPMO Genes in Filamentous Fungi.

Lytic polysaccharide monooxygenases (LPMOs) are mono-copper enzymes that oxidatively degrade various polysaccharides. Genes encoding LPMOs in the AA9 family are abundant in filamentous fungi while their multiplicity remains elusive. We describe a detailed functional characterization of six AA9 LPMOs from the ascomycetous fungus Thermothielavioides terrestris LPH172 (syn. Thielavia terrestris). These six LPMOs were shown to be upregulated during growth on different lignocellulosic substrates in our previous study. Here, we produced them heterologously in Pichia pastoris and tested their activity on various model and native plant cell wall substrates. All six T. terrestris AA9 (TtAA9) LPMOs produced hydrogen peroxide in the absence of polysaccharide substrate and displayed peroxidase-like activity on a model substrate, yet only five of them were active on selected cellulosic substrates. TtLPMO9A and TtLPMO9E were also active on birch acetylated glucuronoxylan, but only when the xylan was combined with phosphoric acid-swollen cellulose (PASC). Another of the six AA9s, TtLPMO9G, was active on spruce arabinoglucuronoxylan mixed with PASC. TtLPMO9A, TtLPMO9E, TtLPMO9G, and TtLPMO9T could degrade tamarind xyloglucan and, with the exception of TtLPMO9T, beechwood xylan when combined with PASC. Interestingly, none of the tested enzymes were active on wheat arabinoxylan, konjac glucomannan, acetylated spruce galactoglucomannan, or cellopentaose. Overall, these functional analyses support the hypothesis that the multiplicity of the fungal LPMO genes assessed in this study relates to the complex and recalcitrant structure of lignocellulosic biomass. Our study also highlights the importance of using native substrates in functional characterization of LPMOs, as we were able to demonstrate distinct, previously unreported xylan-degrading activities of AA9 LPMOs using such substrates. IMPORTANCE The discovery of LPMOs in 2010 has revolutionized the industrial biotechnology field, mainly by increasing the efficiency of cellulolytic enzyme cocktails. Nonetheless, the biological purpose of the multiplicity of LPMO-encoding genes in filamentous fungi has remained an open question. Here, we address this point by showing that six AA9 LPMOs from a single fungal strain have various substrate preferences and activities on tested cellulosic and hemicellulosic substrates, including several native xylan substrates. Importantly, several of these activities could only be detected when using copolymeric substrates that likely resemble plant cell walls more than single fractionated polysaccharides do. Our results suggest that LPMOs have evolved to contribute to the degradation of different complex structures in plant cell walls where different biomass polymers are closely associated. This knowledge together with the elucidated novel xylanolytic activities could aid in further optimization of enzymatic cocktails for efficient degradation of lignocellulosic substrates and more.

Structural Alterations of Mucins Are Associated with Losses in Functionality.

Commercial mucin glycoproteins are routinely used as a model to investigate the broad range of important functions mucins fulfill in our bodies, including lubrication, protection against hostile germs, and the accommodation of a healthy microbiome. Moreover, purified mucins are increasingly selected as building blocks for multifunctional materials, i.e., as components of hydrogels or coatings. By performing a detailed side-by-side comparison of commercially available and lab-purified variants of porcine gastric mucins, we decipher key molecular motifs that are crucial for mucin functionality. As two main structural features, we identify the hydrophobic termini and the hydrophilic glycosylation pattern of the mucin glycoprotein; moreover, we describe how alterations in those structural motifs affect the different properties of mucins-on both microscopic and macroscopic levels. This study provides a detailed understanding of how distinct functionalities of gastric mucins are established, and it highlights the need for high-quality mucins-for both basic research and the development of mucin-based medical products.

Glycan Residues Balance Analysis - GReBA: A novel model for the N-linked glycosylation of IgG produced by CHO cells.

The structure of N-linked glycosylation is a very important quality attribute for therapeutic monoclonal antibodies. Different carbon sources in cell culture media, such as mannose and galactose, have been reported to have different influences on the glycosylation patterns. Accurate prediction and control of the glycosylation profile are important for the process development of mammalian cell cultures. In this study, a mathematical model, that we named Glycan Residues Balance Analysis (GReBA), was developed based on the concept of Elementary Flux Mode (EFM), and used to predict the glycosylation profile for steady state cell cultures. Experiments were carried out in pseudo-perfusion cultivation of antibody producing Chinese Hamster Ovary (CHO) cells with various concentrations and combinations of glucose, mannose and galactose. Cultivation of CHO cells with mannose or the combinations of mannose and galactose resulted in decreased lactate and ammonium production, and more matured glycosylation patterns compared to the cultures with glucose. Furthermore, the growth rate and IgG productivity were similar in all the conditions. When the cells were cultured with galactose alone, lactate was fed as well to be used as complementary carbon source, leading to cell growth rate and IgG productivity comparable to feeding the other sugars. The data of the glycoprofiles were used for training the model, and then to simulate the glycosylation changes with varying the concentrations of mannose and galactose. In this study we showed that the GReBA model had a good predictive capacity of the N-linked glycosylation. The GReBA can be used as a guidance for development of glycoprotein cultivation processes.

Influence of Solubility on the Adsorption of Different Xyloglucan Fractions at Cellulose-Water Interfaces.

Xylogucan (XG) fractions with different molar masses were prepared while preserving the natural structure of the XG. The solubility of the fractions was investigated using light scattering, chromatography, and microscopy techniques. The conformational changes of the XG molecules and their association and phase separation were investigated together with concentration and molar mass changes. The knowledge gained was then applied to investigate the interaction of different XG fractions at cellulose model surfaces using a quartz crystal microbalance with dissipation. The results indicate that there is a cluster formation and phase separation of the XG molecules at the cellulose/water interface induced by the increase in XG concentration close to the surface. Concomitantly, the adsorption regimes are altered for the XG fractions depending on the solubility properties, indicating that the insolubility, association, and phase separation of XGs in aqueous media affect their interaction with cellulose. The study is of vital importance for improving the functionality of sustainable materials made from xyloglucan/cellulose natural composites.

Focused Metabolism of ß-Glucans by the Soil Bacteroidetes Species Chitinophaga pinensis.

The genome and natural habitat of Chitinophaga pinensis suggest it has the ability to degrade a wide variety of carbohydrate-based biomass. Complementing our earlier investigations into the hydrolysis of some plant polysaccharides, we now show that C. pinensis can grow directly on spruce wood and on the fungal fruiting body. Growth was stronger on fungal material, although secreted enzyme activity was high in both cases, and all biomass-induced secretomes showed a predominance of ß-glucanase activities. We therefore conducted a screen for growth on and hydrolysis of ß-glucans isolated from different sources. Most noncrystalline ß-glucans supported good growth, with variable efficiencies of polysaccharide deconstruction and oligosaccharide uptake, depending on the polysaccharide backbone linkage. In all cases, ß-glucan was the only type of polysaccharide that was effectively hydrolyzed by secreted enzymes. This contrasts with the secretion of enzymes with a broad range of activities observed during growth on complex heteroglycans. Our findings imply a role for C. pinensis in the turnover of multiple types of biomass and suggest that the species may have two metabolic modes: a "scavenging mode," where multiple different types of glycan may be degraded, and a more "focused mode" of ß-glucan metabolism. The significant accumulation of some types of ß-gluco-oligosaccharides in growth media may be due to the lack of an appropriate transport mechanism, and we propose that this is due to the specificity of expressed polysaccharide utilization loci. We present a hypothetical model for ß-glucan metabolism by C. pinensis that suggests the potential for nutrient sharing among the microbial litter community.IMPORTANCE It is well known that the forest litter layer is inhabited by a complex microbial community of bacteria and fungi. However, while the importance of fungi in the turnover of natural biomass is well established, the role of their bacterial counterparts is less extensively studied. We show that Chitinophaga pinensis, a prominent member of an important bacterial genus, is capable of using both plant and fungal biomass as a nutrient source but is particularly effective at deconstructing dead fungal material. The turnover of dead fungus is key in natural elemental cycles in the forest. We show that C. pinensis can perform extensive degradation of this material to support its own growth while also releasing sugars that may serve as nutrients for other microbial species. Our work adds detail to an increasingly complex picture of life among the environmental microbiota.

Experimental and Theoretical Evaluation of the Solubility/Insolubility of Spruce Xylan (Arabino Glucuronoxylan).

The molecular solubility of softwood arabinoglucuronoxylan (AGX) has been thoroughly investigated, and it has been shown that the chemical and physical structures of the extracted hemicellulose are not significantly influenced by different purification steps, but a transient molecular solubility of AGX was observed in aqueous media at low concentrations (1 g/L) when the dissolved macromolecules had a hydrodynamic diameter of up to 10 nm. A phase separation was detected when the concentration was increased to 15 g/L leading to an association of the smaller molecules into fractal structures with a considerably larger diameter, even though the dispersions were still transparent to ocular inspection. Dynamic Light Scattering and Cryo-Transmission Electron Microscopy showed dimensions in the range of 1000 nm. The phase separation of the sample was further characterized by estimating the χ-interaction parameter of AGX in water using the Flory-Huggins theory, and the results supported that water is a poor solvent for AGX. This behavior is crucial when films and hydrogels based on these biopolymers are made, since the association will dramatically affect barrier and mechanical properties of films made from these materials.

Gelatin-Based Antimicrobial Films Incorporating Pomegranate (Punica granatum L.) Seed Juice by-Product.

Pomegranate (Punica granatum L.) seed juice by-product (PSP) was added as reinforcing and antimicrobial agent to fish gelatin (FG) films as a promising eco-friendly active material for food packaging applications. A complete linkage analysis of polysaccharides in PSP showed xylan and cellulose as main components. This residue showed also high total phenolic content and antioxidant activity. Three formulations were processed by adding PSP to FG (0, 10, 30 wt. %) by the casting technique, showing films with 10 wt. % of PSP the best performance. The addition of PSP decreased elongation at break and increased stiffness in the FG films, particularly for 30 wt. % loading. A good compatibility between FG and PSP was observed by SEM. No significant (p < 0.05) differences were obtained for barrier properties to oxygen and water vapour permeability compared to the control with the incorporation of PSP, whereas water resistance considerably increased and transparency values decreased (p < 0.05). High thermal stability of films and inhibition against S. aureus were observed. The addition of PSP at 10 wt. % into FG was shown as a potential strategy to maintain the integrity of the material and protect food against lipid oxidation, reducing huge amounts of pomegranate and fish wastes.

Regular Motifs in Xylan Modulate Molecular Flexibility and Interactions with Cellulose Surfaces.

Xylan is tightly associated with cellulose and lignin in secondary plant cell walls, contributing to its rigidity and structural integrity in vascular plants. However, the molecular features and the nanoscale forces that control the interactions among cellulose microfibrils, hemicelluloses, and lignin are still not well understood. Here, we combine comprehensive mass spectrometric glycan sequencing and molecular dynamics simulations to elucidate the substitution pattern in softwood xylans and to investigate the effect of distinct intramolecular motifs on xylan conformation and on the interaction with cellulose surfaces in Norway spruce (Picea abies). We confirm the presence of motifs with evenly spaced glycosyl decorations on the xylan backbone, together with minor motifs with consecutive glucuronation. These domains are differently enriched in xylan fractions extracted by alkali and subcritical water, which indicates their preferential positioning in the secondary plant cell wall ultrastructure. The flexibility of the 3-fold screw conformation of xylan in solution is enhanced by the presence of arabinofuranosyl decorations. Additionally, molecular dynamic simulations suggest that the glycosyl substitutions in xylan are not only sterically tolerated by the cellulose surfaces but that they increase the affinity for cellulose and favor the stabilization of the 2-fold screw conformation. This effect is more significant for the hydrophobic surface compared with the hydrophilic ones, which demonstrates the importance of nonpolar driving forces on the structural integrity of secondary plant cell walls. These novel molecular insights contribute to an improved understanding of the supramolecular architecture of plant secondary cell walls and have fundamental implications for overcoming lignocellulose recalcitrance and for the design of advanced wood-based materials.

The ß-1,3-glucanosyltransferases (Gels) affect the structure of the rice blast fungal cell wall during appressorium-mediated plant infection.

The fungal wall is pivotal for cell shape and function, and in interfacial protection during host infection and environmental challenge. Here, we provide the first description of the carbohydrate composition and structure of the cell wall of the rice blast fungus Magnaporthe oryzae. We focus on the family of glucan elongation proteins (Gels) and characterize five putative ß-1,3-glucan glucanosyltransferases that each carry the Glycoside Hydrolase 72 signature. We generated targeted deletion mutants of all Gel isoforms, that is, the GH72+ , which carry a putative carbohydrate-binding module, and the GH72- Gels, without this motif. We reveal that M. oryzae GH72+ GELs are expressed in spores and during both infective and vegetative growth, but each individual Gel enzymes are dispensable for pathogenicity. Further, we demonstrated that a Δgel1Δgel3Δgel4 null mutant has a modified cell wall in which 1,3-glucans have a higher degree of polymerization and are less branched than the wild-type strain. The mutant showed significant differences in global patterns of gene expression, a hyper-branching phenotype and no sporulation, and thus was unable to cause rice blast lesions (except via wounded tissues). We conclude that Gel proteins play significant roles in structural modification of the fungal cell wall during appressorium-mediated plant infection.

Solubility of Softwood Hemicelluloses.

It is demonstrated that the molecular solubility of softwood hemicelluloses is significantly influenced by pretreatment of the fibers, extraction, and downstream processing. To quantify these effects, four hemicellulose samples were extracted from different thermomechanical pulps of Norway spruce. The molecular solubility of the samples was characterized by size and molar mass distributions, and the morphology of the molecules was studied using high resolution microscopy techniques. All extracted samples were well dispersed in aqueous media creating transparent dispersions, but dynamic light scattering measurements showed that molecular solubility can only be achieved using specific pretreatments and extractions. The procedure yields acetylated galactoglucomannan (AcGGM)-rich hemicelluloses with an average molar mass of 21-35 kDa and a diameter up to 10 nm but also shows that water is a poor solvent for this sample since an association is detected as soon as the concentration is about 20 g/L. These associated hemicellulose dispersions are still absolutely clear on visual inspection, underlining the need for careful measurement when assessing the solubility of wood hemicelluloses.

A molecular dynamics study of the effect of glycosidic linkage type in the hemicellulose backbone on the molecular chain flexibility.

The macromolecular conformation of the constituent polysaccharides in lignocellulosic biomass influences their supramolecular interactions, and therefore their function in plants and their performance in technical products. The flexibility of glycosidic linkages from the backbone of hemicelluloses was studied by evaluating the conformational freedom of the φ and ψ dihedral angles using molecular dynamic simulations, additionally selected molecules were correlated with experimental data by nuclear magnetic resonance spectroscopy. Three types of ß-(1→4) glycosidic linkages involving the monosaccharides (Glcp, Xylp and Manp) present in the backbone of hemicelluloses were defined. Different di- and tetrasaccharides with combinations of such sugar monomers from hemicelluloses were simulated, and free energy maps of the φ - ψ space and hydrogen-bonding patterns were obtained. The glycosidic linkage between Glc-Glc or Glc-Man (C-type) was the stiffest with mainly one probable conformation; the linkage from Man-Man or Man-Glc (M-type) was similar but with an increased probability for an alternative conformation making it more flexible, and the linkage between two Xyl-units (X-type) was the most flexible with two almost equally populated conformations. Glycosidic linkages of the same type showed essentially the same conformational space in both disaccharides and in the central region of tetrasaccharides. Different probabilities of glycosidic linkage conformations in the backbone of hemicelluloses can be directly estimated from the free energy maps, which to a large degree affect the overall macromolecular conformations of these polymers. The information gained contributes to an increased understanding of the function of hemicelluloses both in the cell wall and in technical products.

Analytical markers for silk degradation: comparing historic silk and silk artificially aged in different environments.

Suitable analytical markers to assess the degree of degradation of historic silk textiles at molecular and macroscopic levels have been identified and compared with silk textiles aged artificially in different environments, namely (i) ultraviolet (UV) exposure, (ii) thermo-oxidation, (iii) controlled humidity and (iv) pH. The changes at the molecular level in the amino acid composition, the formation of oxidative moieties, crystallinity and molecular weight correlate well with the changes in the macroscopic properties such as brightness, pH and mechanical properties. These analytical markers are useful to understand the degradation mechanisms that silk textiles undergo under different degradation environments, involving oxidation processes, hydrolysis, chain scission and physical arrangements. Thermo-oxidation at high temperatures proves to be the accelerated ageing procedure producing silk samples that most resembled the degree of degradation of early seventeenth-century silk. These analytical markers will be valuable to support the textile conservation tasks currently being performed in museums to preserve our heritage.

CysK2 from Mycobacterium tuberculosis is an O-phospho-L-serine-dependent S-sulfocysteine synthase.

Mycobacterium tuberculosis is dependent on cysteine biosynthesis, and reduced sulfur compounds such as mycothiol synthesized from cysteine serve in first-line defense mechanisms against oxidative stress imposed by macrophages. Two biosynthetic routes to l-cysteine, each with its own specific cysteine synthase (CysK1 and CysM), have been described in M. tuberculosis, but the function of a third putative sulfhydrylase in this pathogen, CysK2, has remained elusive. We present biochemical and biophysical evidence that CysK2 is an S-sulfocysteine synthase, utilizing O-phosphoserine (OPS) and thiosulfate as substrates. The enzyme uses a mechanism via a central aminoacrylate intermediate that is similar to that of other members of this pyridoxal phosphate-dependent enzyme family. The apparent second-order rate of the first half-reaction with OPS was determined as kmax/Ks = (3.97 × 10(3)) ± 619 M(-1) s(-1), which compares well to the OPS-specific mycobacterial cysteine synthase CysM with a kmax/Ks of (1.34 × 10(3)) ± 48.2. Notably, CysK2 does not utilize thiocarboxylated CysO as a sulfur donor but accepts thiosulfate and sulfide as donor substrates. The specificity constant kcat/Km for thiosulfate is 40-fold higher than for sulfide, suggesting an annotation as S-sulfocysteine synthase. Mycobacterial CysK2 thus provides a third metabolic route to cysteine, either directly using sulfide as donor or indirectly via S-sulfocysteine. Hypothetically, S-sulfocysteine could also act as a signaling molecule triggering additional responses in redox defense in the pathogen upon exposure to reactive oxygen species during dormancy.

Enzymatic characterization of a glycoside hydrolase family 5 subfamily 7 (GH5_7) mannanase from Arabidopsis thaliana.

Each plant genome contains a repertoire of ß-mannanase genes belonging to glycoside hydrolase family 5 subfamily 7 (GH5_7), putatively involved in the degradation and modification of various plant mannan polysaccharides, but very few have been characterized at the gene product level. The current study presents recombinant production and in vitro characterization of AtMan5-1 as a first step towards the exploration of the catalytic capacity of Arabidopsis thaliana ß-mannanase. The target enzyme was expressed in both E. coli (AtMan5-1e) and P. pastoris (AtMan5-1p). The main difference between the two forms was a higher observed thermal stability for AtMan5-1p, presumably due to glycosylation of that particular variant. AtMan5-1 displayed optimal activity at pH 5 and 35 °C and hydrolyzed polymeric carob galactomannan, konjac glucomannan, and spruce galactoglucomannan as well as oligomeric mannopentaose and mannohexaose. However, the galactose-rich and highly branched guar gum was not as efficiently degraded. AtMan5-1 activity was enhanced by Co(2+) and inhibited by Mn(2+). The catalytic efficiency values for carob galactomannan were 426.8 and 368.1 min(-1) mg(-1) mL for AtMan5-1e and AtMan5-1p, respectively. Product analysis of AtMan5-1p suggested that at least five substrate-binding sites were required for manno-oligosaccharide hydrolysis, and that the enzyme also can act as a transglycosylase.