Synthesis of a natural core substrate with lignin-xylan cross-linkage for unveiling the productive kinetic parameters of glucuronoyl esterase.

Lignin-carbohydrate complexes (LCCs) present a considerable hurdle to the economic utilization of lignocellulosic biomass. Glucuronoyl esterase (GE) is an LCC-degrading enzyme that catalyzes the cleavage of the cross-linkages between lignin and xylan in LCCs. Benzyl-d-glucuronate (Bn-GlcA), a commercially available substrate, is widely used to evaluate GE activity assays. However, since Bn-GlcA lacks the structural backbone of naturally occurring LCCs, the mechanisms underlying the activity of GEs and their diversity in the structure-activity relationship are not fully understood. Herein, we provided a synthesis scheme for designing 1,23-α-d-(6-benzyl-4-O-methyl-glucuronyl)-1,4-ß-d-xylotriose (Bn-MeGlcA3Xyl3) as a natural core substrate bearing cross-linkage between lignin and glucuronoxylan. A well-defined and yet more realistic synthetic substrate was successfully synthesized via a key step of the benzyl esterification of 4-O-methyl-glucuronyl-1,4-ß-d-xylotriose (MeGlcA3Xyl3), a minimized fragment of glucuronoxylan enzymatically digested by ß-1,4-xylanase. To the best of our knowledge, this is the first report of the productive GE kinetic analysis using this substrate. Kinetic parameters of the GE from the fungal Pestalotiopsis sp. AN-7 (PesGE), i.e., the Km, Vmax, and kcat of Bn-MeGlcA3Xyl3, were 0.43 mM, 55.5 µmol min-1·mg-1, and 35.8 s-1, respectively. On the other hand, as reported to date, the productive kinetic parameters for Bn-GlcA were not obtained because of its excessively high Km value (>16 mM). The substantial variance in the enzymatic activity of PesGE regarding substrate-binding affinity between Bn-MeGlcA3Xyl3 and Bn-GlcA was also demonstrated using in silico docking simulation. These results suggested that the extended xylan fragment is a key structural determinant affecting PesGE's substrate recognition. Furthermore, the presence of a natural xylan backbone allows for evaluating the enzyme activity of xylan-degrading enzymes. Accordingly, the synthesized substrate with the natural core structure of LCC allowed us to unveil the productive kinetic parameters of GEs, serving as a versatile substrate for further elucidating the cascade reaction of GE and xylan-degrading enzymes.

Distribution of xylan linked glucuronic acid labelled by molecularly imprinted polymers on pulp fiber surface.

Efficiently utilization of plant resources is heavily restricted by the resistance of lignocellulose in plant cells, which is related to the interlinkages of lignocellulose components. Hemicellulose in plant cell wall is bound to cellulose by hydrogen bond and linked with lignin in lignin-carbohydrate complex (LCC). In the xylan chain of hemicellulose, glucuronic acid (GA) is a typical side-group, which provides clues for us to label and locate hemicellulose. The way to label GA on the surface of pulp fibers obtained from pulping process is benefit to explore the deconstruction of lignocellulose. Herein, a new visualization method, fluorescence modified molecularly imprinted polymers (MIP) were applied to recognize and locate GA on the pulp fiber surface. The method combining fluorescence imaging and integrated 3D fiber structure verified the feasibility of the MIP for specific GA recognition. The results showed that xylan (represented by GA) was closely attached to lignin, distributed along the inner wall of pulp fiber cells, and gradually taken off from the inside edge of fiber cells with the deconstruction of lignocellulose. This research provided a basis to develop visualization bioimaging technology to identify biomass components.

Identification of glycosyltransferases mediating 2-O-arabinopyranosyl and 2-O-galactosyl substitutions of glucuronosyl side chains of xylan.

Xylan is one of the major hemicelluloses in plant cell walls and its xylosyl backbone is often decorated at O-2 with glucuronic acid (GlcA) and/or methylglucuronic acid (MeGlcA) residues. The GlcA/MeGlcA side chains may be further substituted with 2-O-arabinopyranose (Arap) or 2-O-galactopyranose (Gal) residues in some plant species, but the enzymes responsible for these substitutions remain unknown. During our endeavor to investigate the enzymatic activities of Arabidopsis MUR3-clade members of the GT47 glycosyltransferase family, we found that one of them was able to transfer Arap from UDP-Arap onto O-2 of GlcA side chains of xylan, and thus it was named xylan 2-O-arabinopyranosyltransferase 1 (AtXAPT1). The function of AtXAPT1 was verified in planta by its T-DNA knockout mutation showing a loss of the Arap substitution on xylan GlcA side chains. Further biochemical characterization of XAPT close homologs from other plant species demonstrated that while the poplar ones had the same catalytic activity as AtXAPT1, those from Eucalyptus, lemon-scented gum, sea apple, 'Ohi'a lehua, duckweed and purple yam were capable of catalyzing both 2-O-Arap and 2-O-Gal substitutions of xylan GlcA side chains albeit with differential activities. Sequential reactions with XAPTs and glucuronoxylan methyltransferase 3 (GXM3) showed that XAPTs acted poorly on MeGlcA side chains, whereas GXM3 could efficiently methylate arabinosylated or galactosylated GlcA side chains of xylan. Furthermore, molecular docking and site-directed mutagenesis analyses of Eucalyptus XAPT1 revealed critical roles of several amino acid residues at the putative active site in its activity. Together, these findings establish that XAPTs residing in the MUR3 clade of family GT47 are responsible for 2-O-arabinopyranosylation and 2-O-galactosylation of GlcA side chains of xylan.

Structure and rheology of the polysaccharides from Schizymenia dubyi: Isolation of an unusual glucurono-carrageenan.

The system of polysaccharides from Schizymenia dubyi (Nemastomatales) was investigated. It contains a mixture of hybrid dl galactans (SH-S) and carrageenan-like polysaccharides, which were separated by means of precipitation with KCl at high concentrations. The structural features of the carrageenan-like fraction (SH-I) were investigated by methylation analysis, desulfation, uronic acid reduction, and NMR spectroscopy. It was concluded that the structure has the typical alternance α-(1 â†’ 3), ß-(1 â†’ 4) of d-galactose units, with most of the 3-linked units sulfated in O-2 (and some in O-4), and most of the 4-linked units sulfated in O-3, and substituted in O-2 by single stubs of ß-d-glucuronic acid (partly sulfated in each of the three available positions). This substituent has been only seldom found in red seaweed galactans. Rheological studies of 5 % and 10 % w/v SH, SH-S and SH-I aqueous systems, either without ions, or in KCl or CaCl2 solution gave thickening behaviors. Their random coil conformations justify the pseudoplastic behavior observed in the viscosity versus shear rate curves. As SH-S and SH-I are both contained in SH, an interpenetrating network could form in SH between the glucurono-carrageenan and the agaran, as inferred from the mechanical spectra recorded in water, especially with potassium ion.

Glucuronic acid confers colonization advantage to enteric pathogens.

Glucuronidation is a detoxification process to eliminate endo- and xeno-biotics and neurotransmitters from the host circulation. Glucuronosyltransferase binds these compounds to glucuronic acid (GlcA), deactivating them and allowing their elimination through the gastrointestinal (GI) tract. However, the microbiota produces ß-glucuronidases that release GlcA and reactivate these compounds. Enteric pathogens such as enterohemorrhagic Escherichia coli (EHEC) and Citrobacter rodentium sense and utilize galacturonic acid (GalA), an isomer of GlcA, to outcompete the microbiota promoting gut colonization. However, the role of GlcA in pathogen colonization has not been explored. Here, we show that treatment of mice with a microbial ß-glucuronidase inhibitor (GUSi) decreased C. rodentium's colonization of the GI tract, without modulating bacterial virulence or host inflammation. Metagenomic studies indicated that GUSi did not change the composition of the intestinal microbiota in these animals. GlcA confers an advantage for pathogen expansion through its utilization as a carbon source. Congruently mutants unable to catabolize GlcA depict lower GI colonization compared to wild type and are not sensitive to GUSi. Germfree mice colonized with a commensal E. coli deficient for ß-glucuronidase production led to a decrease of C. rodentium tissue colonization, compared to animals monocolonized with an E. coli proficient for production of this enzyme. GlcA is not sensed as a signal and doesn't activate virulence expression but is used as a metabolite. Because pathogens can use GlcA to promote their colonization, inhibitors of microbial ß-glucuronidases could be a unique therapeutic against enteric infections without disturbing the host or microbiota physiology.

Metabolomics analysis reveals novel serum metabolite alterations in cancer cachexia.

Background: Cachexia is a body wasting syndrome that significantly affects well-being and prognosis of cancer patients, without effective treatment. Serum metabolites take part in pathophysiological processes of cancer cachexia, but apart from altered levels of select serum metabolites, little is known on the global changes of the overall serum metabolome, which represents a functional readout of the whole-body metabolic state. Here, we aimed to comprehensively characterize serum metabolite alterations and analyze associated pathways in cachectic cancer patients to gain new insights that could help instruct strategies for novel interventions of greater clinical benefit. Methods: Serum was sampled from 120 metastatic cancer patients (stage UICC IV). Patients were grouped as cachectic or non-cachectic according to the criteria for cancer cachexia agreed upon international consensus (main criterium: weight loss adjusted to body mass index). Samples were pooled by cachexia phenotype and assayed using non-targeted gas chromatography-mass spectrometry (GC-MS). Normalized metabolite levels were compared using t-test (p < 0.05, adjusted for false discovery rate) and partial least squares discriminant analysis (PLS-DA). Machine-learning models were applied to identify metabolite signatures for separating cachexia states. Significant metabolites underwent MetaboAnalyst 5.0 pathway analysis. Results: Comparative analyses included 78 cachectic and 42 non-cachectic patients. Cachectic patients exhibited 19 annotable, significantly elevated (including glucose and fructose) or decreased (mostly amino acids) metabolites associating with aminoacyl-tRNA, glutathione and amino acid metabolism pathways. PLS-DA showed distinct clusters (accuracy: 85.6%), and machine-learning models identified metabolic signatures for separating cachectic states (accuracy: 83.2%; area under ROC: 88.0%). We newly identified altered blood levels of erythronic acid and glucuronic acid in human cancer cachexia, potentially linked to pentose-phosphate and detoxification pathways. Conclusion: We found both known and yet unknown serum metabolite and metabolic pathway alterations in cachectic cancer patients that collectively support a whole-body metabolic state with impaired detoxification capability, altered glucose and fructose metabolism, and substrate supply for increased and/or distinct metabolic needs of cachexia-associated tumors. These findings together imply vulnerabilities, dependencies and targets for novel interventions that have potential to make a significant impact on future research in an important field of cancer patient care.

Strategies for the biological synthesis of D-glucuronic acid and its derivatives.

D-glucuronic acid is a kind of glucose derivative, which has excellent properties such as anti-oxidation, treatment of liver disease and hyperlipidemia, and has been widely used in medicine, cosmetics, food and other fields. The traditional production methods of D-glucuronic acid mainly include natural extraction and chemical synthesis, which can no longer meet the growing market demand. The production of D-glucuronic acid by biocatalysis has become a promising alternative method because of its high efficiency and environmental friendliness. This review describes different production methods of D-glucuronic acid, including single enzyme catalysis, multi-enzyme cascade, whole cell catalysis and co-culture, as well as the intervention of some special catalysts. In addition, some feasible enzyme engineering strategies are provided, including the application of enzyme immobilized scaffold, enzyme mutation and high-throughput screening, which provide good ideas for the research of D-glucuronic acid biocatalysis.

Stereocontrolled Synthesis and Conformational Analysis of a Series of Disaccharides α,ß-d-GlcA-(1→3)-α-L-Fuc.

D-Glucuronic acid is a fundamental building block of many biologically important polysaccharides, either in its non-substituted form or bearing a variety of substituents, among them sulfates. We have previously performed a study of the effects of exhaustive sulfation on the conformational behavior of ß-gluronopyranosides. Herein, we report an investigation comparing α- and ß-derivatives of this monosaccharide within the title disaccharides using NMR and quantum chemistry approaches. It was found that for α-linked disaccharides, the introduction of sulfates did not greatly affect their conformational behavior. However, for ß-derivatives, considerable conformational changes were observed. In general, they resemble those that took place for the monosaccharides, except that NOESY experiments and calculations of intra-ring spin-spin coupling constants suggest the presence of a 1S5 conformer along with 3S1 in the fully sulfated disaccharide. During the synthesis of model compounds, hydrogen bond-mediated aglycone delivery was used as an α-directing stereocontrol approach in the glucuronidation reaction.

Expanding the synthesis of a library of potent glucuronic acid glycodendrons for Dengue virus inhibition.

Multivalent glycodendrons are valuable tools to mimic many structural and functional features of cell-surface glycoconjugates and its focal position scaffolds represent important components to increase specificity and affinity. Previous work in our group described the preparation of a tetravalent glucuronic acid dendron that binds with good affinity to Dengue virus envelope protein (KD = 22 µM). Herein, the chemical synthesis and binding analysis of a new library of potent glucuronic acid dendrons bearing different functional group at the focal position and different level of multivalency are described. Their chemical synthesis was performed sequentially in three stages and with good yields. Namely a) the chemical synthesis of the oligo and polyalkynyl scaffolds, b) assembling with fully protected glucuronic acid-based azide units by using a microwave assisted copper-catalysed azide-alkyne cycloaddition reaction and c) sequential deprotection of hydroxyl and carboxylic acid groups. Surface Plasmon Resonance studies have demonstrated that the valency and the focal position functional group exert influence on the interaction with Dengue virus envelope protein. Molecular modelling studies were carried out in order to understand the binding observed. This work reports an efficient glycodendrons chemical synthesis that provides appropriate focal position functional group and multivalence, that offer an easy and versatile strategy to find new active compounds against Dengue virus.

Glucuronic acid is a novel source of pentosidine, associated with schizophrenia.

Pentosidine (PEN) is an advanced glycation end-product (AGEs), where a fluorescent cross-link is formed between lysine and arginine residues in proteins. Accumulation of PEN is associated with aging and various diseases. We previously reported that a subpopulation of patients with schizophrenia showed PEN accumulation in the blood, having severe clinical features. PEN is thought to be produced from glucose, fructose, pentoses, or ascorbate. However, patients with schizophrenia with high PEN levels present no elevation of these precursors of PEN in their blood. Therefore, the molecular mechanisms underlying PEN accumulation and the molecular pathogenesis of schizophrenia associated with PEN accumulation remain unclear. Here, we identified glucuronic acid (GlcA) as a novel precursor of PEN from the plasma of subjects with high PEN levels. We demonstrated that PEN can be generated from GlcA, both in vitro and in vivo. Furthermore, we found that GlcA was associated with the diagnosis of schizophrenia. Among patients with high PEN, the proportion of those who also have high GlcA is 25.6%. We also showed that Aldo-keto reductase (AKR) activity to degrade GlcA was decreased in patients with schizophrenia, and its activity was negatively correlated with GlcA levels in the plasma. This is the first report to show that PEN is generated from GlcA. In the future, this finding will contribute to understanding the molecular pathogenesis of not only schizophrenia but also other diseases with PEN accumulation.