Use of xylosidase 3C from Segatella baroniae to discriminate xylan non-reducing terminus substitution characteristics.

OBJECTIVE: New characterized carbohydrate-active enzymes are needed for use as tools to discriminate complex carbohydrate structural features. Fungal glycoside hydrolase family 3 (GH3) ß-xylosidases have been shown to be useful for the structural elucidation of glucuronic acid (GlcA) and arabinofuranose (Araf) substituted oligoxylosides. A homolog of these GH3 fungal enzymes from the bacterium Segatella baroniae (basonym Prevotella bryantii), Xyl3C, has been previously characterized, but those studies did not address important functional specificity features. In an interest to utilize this enzyme for laboratory methods intended to discriminate the structure of the non-reducing terminus of substituted xylooligosaccharides, we have further characterized this GH3 xylosidase. RESULTS: In addition to verification of basic functional characteristics of this xylosidase we have determined its mode of action as it relates to non-reducing end xylose release from GlcA and Araf substituted oligoxylosides. Xyl3C cleaves xylose from the non-reducing terminus of ß-1,4-xylan until occurrence of a penultimate substituted xylose. If this substitution is O2 linked, then Xyl3C removes the non-reducing xylose to leave the substituted xylose as the new non-reducing terminus. However, if the substitution is O3 linked, Xyl3C does not hydrolyze, thus leaving the substitution one-xylose (penultimate) from the non-reducing terminus. Hence, Xyl3C enables discrimination between O2 and O3 linked substitutions on the xylose penultimate to the non-reducing end. These findings are contrasted using a homologous enzyme also from S. baroniae, Xyl3B, which is found to yield a penultimate substituted nonreducing terminus regardless of which GlcA or Araf substitution exists.

Sugarcane bagasse derived xylooligosaccharides produced by an arabinofuranosidase/xylobiohydrolase from Bifidobacterium longum in synergism with xylanases.

Arabinoxylan is a major hemicellulose in the sugarcane plant cell wall with arabinose decorations that impose steric restrictions on the activity of xylanases against this substrate. Enzymatic removal of the decorations by arabinofuranosidases can allow a more efficient arabinoxylan degradation by xylanases. Here we produced and characterized a recombinant Bifidobacterium longum arabinofuranosidase from glycoside hydrolase family 43 (BlAbf43) and applied it, together with GH10 and GH11 xylanases, to produce xylooligosaccharides (XOS) from wheat arabinoxylan and alkali pretreated sugarcane bagasse. The enzyme synergistically enhanced XOS production by GH10 and GH11 xylanases, being particularly efficient in combination with the latter family of enzymes, with a degree of synergism of 1.7. We also demonstrated that the enzyme is capable of not only removing arabinose decorations from the arabinoxylan and from the non-reducing end of the oligomeric substrates, but also hydrolyzing the xylan backbone yielding mostly xylobiose and xylose in particular cases. Structural studies of BlAbf43 shed light on the molecular basis of the substrate recognition and allowed hypothesizing on the structural reasons of its multifunctionality.

Retrospective Data Analysis Reveals Unusual Metabolism Pattern of Ethanol in Pediatrics as Compared to Adult and Geriatric Populations.

BACKGROUND: About 95% of consumed ethanol is metabolized by oxidative pathways. Less than 1% is metabolized via nonoxidative pathways: glucuronidation, sulfation, and the formation of fatty acid esters of ethanol. In neonates, the glucuronidation pathway has been reported to be underdeveloped but matures with age. This work compared the test results of patients' random urine samples submitted to our facility for ethyl glucuronide (EtG) and ethyl sulfate (EtS) measurements across pediatric and adult populations. METHODS: Test results (n = 63 498) from urine samples tested for EtG and EtS by quantitative liquid chromatography-tandem mass spectrometry at our facility were utilized for this study. EtG and EtS concentrations were compared across the age partitions 0 to 17 years (pediatric), 18 to 80 years (adult), and 81 to 100 years (geriatric). Eight pediatric patients from a tertiary academic hospital contributed clinical context via abstracted clinical information. RESULTS: Across the individual age partitions, 60% to 65% of patients had both EtG and EtS present in urine. Approximately 5% to 10% of patients had only EtG, and 25% to 35% had neither metabolite present. The lowest percentages (<1.5%) had EtS present in the absence of EtG. Markedly, no pediatric patients had only EtS present; compared to the adult population, this was statistically significant (Fisher exact test, P = 0.025). CONCLUSIONS: From the data presented in this work, EtG is more prevalent relative to EtS in urine samples of patients assessed for ethanol exposure.

Heteroxylan hydrolysis by a recombinant cellulase-free GH10 xylanase from the alkaliphilic bacterium Halalkalibacterium halodurans C-125.

The search for affordable enzymes with exceptional characteristics is fundamental to overcoming industrial and environmental constraints. In this study, a recombinant GH10 xylanase (Xyn10-HB) from the extremely alkaliphilic bacterium Halalkalibacterium halodurans C-125 cultivated at pH 10 was cloned and expressed in E. coli BL21(DE3). Removal of the signal peptide improved the expression, and an overall activity of 8 U/mL was obtained in the cell-free supernatant. The molecular weight of purified Xyn10-HB was estimated to be 42.6 kDa by SDS-PAGE. The enzyme was active across a wide pH range (5-10) with optimal activity recorded at pH 8.5 and 60 °C. It also presented good stability with a half-life of 3 h under these conditions. Substrate specificity studies showed that Xyn10-HB is a cellulase-free enzyme that conventionally hydrolyse birchwood and oat spelts xylans (Apparent Km of 0.46 mg/mL and 0.54 mg/mL, respectively). HPLC analysis showed that both xylans hydrolysis produced xylooligosaccharides (XOS) with a degree of polymerization (DP) ranging from 2 to 9. The conversion yield was 77% after 24 h with xylobiose and xylotriose as the main end-reaction products. When assayed on alkali-extracted wheat straw heteroxylan, the Xyn10-HB produced active XOS with antioxidant activity determined by the DPPH radical scavenging method (IC50 of 0.54 mg/mL after 4 h). Owing to its various characteristics, Xyn10-HB xylanase is a promising candidate for multiple biotechnological applications.

Two-domain GH30 xylanase from human gut microbiota as a tool for enzymatic production of xylooligosaccharides: Crystallographic structure and a synergy with GH11 xylosidase.

Production of value-added compounds and sustainable materials from agro-industrial residues is essential for better waste management and building of circular economy. This includes valorization of hemicellulosic fraction of plant biomass, the second most abundant biopolymer from plant cell walls, aiming to produce prebiotic oligosaccharides, widely explored in food and feed industries. In this work, we conducted biochemical and biophysical characterization of a prokaryotic two-domain R. champanellensis xylanase from glycoside hydrolase (GH) family 30 (RcXyn30A), and evaluated its applicability for XOS production from glucuronoxylan in combination with two endo-xylanases from GH10 and GH11 families and a GH11 xylobiohydrolase. RcXyn30A liberates mainly long monoglucuronylated xylooligosaccharides and is inefficient in cleaving unbranched oligosaccharides. Crystallographic structure of RcXyn30A catalytic domain was solved and refined to 1.37 Å resolution. Structural analysis of the catalytic domain releveled that its high affinity for glucuronic acid substituted xylan is due to the coordination of the substrate decoration by several hydrogen bonds and ionic interactions in the subsite -2. Furthermore, the protein has a larger ß5-α5 loop as compared to other GH30 xylanases, which might be crucial for creating an additional aglycone subsite (+3) of the catalytic site. Finally, RcXyn30A activity is synergic to that of GH11 xylobiohydrolase.

Characterization of a novel GH30 non-specific endoxylanase AcXyn30B from Acetivibrio clariflavus.

The xylanolytic enzymes Clocl_1795 and Clocl_2746 from glycoside hydrolase (GH) family 30 are highly abundant in the hemicellulolytic system of Acetivibrio clariflavus (Hungateiclostridium, Clostridium clariflavum). Clocl_1795 has been shown to be a xylobiohydrolase AcXbh30A releasing xylobiose from the non-reducing end of xylan and xylooligosaccharides. In this work, biochemical characterization of Clocl_2746 is presented. The protein, designated AcXyn30B, shows low sequence similarity to other GH30 members and phylogenetic analysis revealed that AcXyn30B and related proteins form a separate clade that is proposed to be a new subfamily GH30_12. AcXyn30B exhibits similar specific activity on glucuronoxylan, arabinoxylan, and aryl glycosides of linear xylooligosaccharides suggesting that it is a non-specific xylanase. From polymeric substrates, it releases the fragments of degrees of polymerization (DP) 2-6. Hydrolysis of different xylooligosaccharides indicates that AcXyn30B requires at least four occupied catalytic subsites for effective cleavage. The ability of the enzyme to hydrolyze a wide range of substrates is interesting for biotechnological applications. In addition to subfamilies GH30_7, GH30_8, and GH30_10, the newly proposed subfamily GH30_12 further widens the spectrum of GH30 subfamilies containing xylanolytic enzymes. KEY POINTS: Bacterial GH30 endoxylanase from A. clariflavus (AcXyn30B) has been characterized AcXyn30B is non-specific xylanase hydrolyzing various xylans and xylooligosaccharides Phylogenetic analysis placed AcXyn30B in a new GH30_12 subfamily.

Influence of chain length on the colonic fermentation of xylooligosaccharides.

Xylooligosaccharides (XOS) have been employed as prebiotics containing oligomers of varying sizes or molecular ratios. XOS with a low degree of polymerization (DP) has been demonstrated to have high prebiotic potential. However, there is limited information regarding the specific chain length of XOS required to elicit distinct responses in the gut microbiota. In this study, we aimed to explore whether variations in XOS DP could alter the fate of colonic fermentation. Five XOS fractions (BWXFs) with DP ranges of >40, 20-40, 10-20, 5-10, and 2-4 were prepared by beechwood xylan autohydrolysis and tested on human gut microbiota. Extracellular XOS degradation was observed for molecules with a DP exceeding 5. BWXF treatments altered the microbial community structures, and substrate size-dependent effects on the microbial composition and metabolic outputs were observed. Bacteroidaceae were specifically enriched by xylan. Lachnospiraceae were particularly stimulated by XOS with a DP of 20-40 and 2-4. Bifidobacteriaceae were notably enriched by XOS with a DP of 5-20. High butyrate yields were obtained from cultures containing long-chain BWXFs. Microbiota responses differed with XOS DP composition changes, and microbial competition with XOS with a DP of 2-4 requires further exploration.

Multi-point immobilization of GH 11 endo-ß-1,4-xylanase on magnetic MOF composites for higher yield of xylo-oligosaccharides.

GH 11 endo-ß-1,4-xylanase (Xy) was a crucial enzyme for xylooligosaccharides (XOS) production. The lower reusability and higher cost of purification has limited the industrial application of Xy. Addressing these challenges, our study utilized various immobilization techniques, different supports and forces for Xy immobilization. This study presents a new method in the development of Fe3O4@PDA@MOF-Xy which is immobilized via multi-point interaction forces, demonstrating a significant advancement in protein loading capacity (80.67 mg/g), and exhibiting remarkable tolerance to acidic and alkaline conditions. This method significantly improved Xy reusability and efficiency for industrial applications, maintaining 60 % activity over 10 cycles. Approximately 23 % XOS production was achieved by Fe3O4@PDA@MOF-Xy. Moreover, the yield of XOS from cobcorn xylan using this system was 1.15 times higher than that of the free enzyme system. These results provide a theoretical and applicative basis for enzyme immobilization and XOS industrial production.

Comprehensive analysis of the UDP-glucuronate decarboxylase (UXS) gene family in tobacco and functional characterization of NtUXS16 in Golgi apparatus in Arabidopsis.

BACKGROUND: UDP-glucuronate decarboxylase (also named UXS) converts UDP-glucuronic acid (UDP-GlcA) to UDP-xylose (UDP-Xyl) by decarboxylation of the C6-carboxylic acid of glucuronic acid. UDP-Xyl is an important sugar donor that is required for the synthesis of plant cell wall polysaccharides. RESULTS: In this study, we first carried out the genome-wide identification of NtUXS genes in tobacco. A total of 17 NtUXS genes were identified, which could be divided into two groups (Group I and II), and the Group II UXSs can be further divided into two subgroups (Group IIa and IIb). Furthermore, the protein structures, intrachromosomal distributions and gene structures were thoroughly analyzed. To experimentally verify the subcellular localization of NtUXS16 protein, we transformed tobacco BY-2 cells with NtUXS16 fused to the monomeric red fluorescence protein (mRFP) at the C terminus under the control of the cauliflower mosaic virus (CaMV) 35S promoter. The fluorescent signals of NtUXS16-mRFP were localized to the medial-Golgi apparatus. Contrary to previous predictions, protease digestion analysis revealed that NtUXS16 is not a type II membrane protein. Overexpression of NtUXS16 in Arabidopsis seedling in darkness led to a significant increase in hypocotyl length and a reduction in root length compared with the wild type. In summary, these results suggest Golgi apparatus localized-NtUXS16 plays an important role in hypocotyl and root growth in the dark. CONCLUSION: Our findings facilitate our understanding of the novel functions of NtUXS16 and provide insights for further exploration of the biological roles of NtUXS genes in tobacco.

UDP-glucuronate metabolism controls RIPK1-driven liver damage in nonalcoholic steatohepatitis.

Hepatocyte apoptosis plays an essential role in the progression of nonalcoholic steatohepatitis (NASH). However, the molecular mechanisms underlying hepatocyte apoptosis remain unclear. Here, we identify UDP-glucose 6-dehydrogenase (UGDH) as a suppressor of NASH-associated liver damage by inhibiting RIPK1 kinase-dependent hepatocyte apoptosis. UGDH is progressively reduced in proportion to NASH severity. UGDH absence from hepatocytes hastens the development of liver damage in male mice with NASH, which is suppressed by RIPK1 kinase-dead knockin mutation. Mechanistically, UGDH suppresses RIPK1 by converting UDP-glucose to UDP-glucuronate, the latter directly binds to the kinase domain of RIPK1 and inhibits its activation. Recovering UDP-glucuronate levels, even after the onset of NASH, improved liver damage. Our findings reveal a role for UGDH and UDP-glucuronate in NASH pathogenesis and uncover a mechanism by which UDP-glucuronate controls hepatocyte apoptosis by targeting RIPK1 kinase, and suggest UDP-glucuronate metabolism as a feasible target for more specific treatment of NASH-associated liver damage.

Role of Bifidobacterium pseudocatenulatum in Degradation and Consumption of Xylan-Derived Carbohydrates.

Xylans, a family of xylose-based polysaccharides, are dietary fibers resistant to digestion. They therefore reach the large intestine intact; there, they are utilized by members of the gut microbiota. They are initially broken down by primary degraders that utilize extracellular xylanases to cleave xylan into smaller oligomers. The resulting xylooligosaccharides (XOS) can either be further metabolized directly by primary degraders or cross-feed secondary consumers, including Bifidobacterium. While several Bifidobacterium species have metabolic systems for XOS, most grow poorly on longer-chain XOS and xylan substrates. In this study, we isolated strains of Bifidobacterium pseudocatenulatum and observed that some, including B. pseudocatenulatum ED02, displayed growth on XOS with a high degree of polymerization (DP) and straight-chain xylan, suggesting a primary degrader phenotype that is rare in Bifidobacterium. In silico analyses revealed that only the genomes of these xylan-fermenting (xylan+) strains contained an extracellular GH10 endo-ß-1.4 xylanase, a key enzyme for primary degradation of xylan. The presence of an extracellular xylanase was confirmed by the appearance of xylan hydrolysis products in cell-free supernatants. Extracellular xylanolytic activity was only detected in xylan+ strains, as indicated by the production of XOS fragments with a DP of 2 to 6, identified by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). Additionally, in vitro fecal fermentations revealed that strains with a xylan+ phenotype can persist with xylan supplementation. These results indicate that xylan+ B. pseudocatenulatum strains may have a competitive advantage in the complex environment of the gastrointestinal tract, due to their ability to act as primary degraders of xylan through extracellular enzymatic degradation. IMPORTANCE The beneficial health effects of dietary fiber are now well established. Moreover, low fiber consumption is associated with increased risks of metabolic and systemic diseases. This so-called "fiber gap" also has a profound impact on the composition of the gut microbiome, leading to a disrupted or dysbiotic microbiota. Therefore, understanding the mechanisms by which keystone bacterial species in the gut utilize xylans and other dietary fibers may provide a basis for developing strategies to restore gut microbiome function. The results described here provide biochemical and genetic evidence for primary xylan utilization by human-derived Bifidobacterium pseudocatenulatum and show also that cooperative utilization of xylans occurs among other members of this species.

Whole Transcriptome Analyses of Apricots and Japanese Plum Fruits after 1-MCP (Ethylene-Inhibitor) and Ethrel (Ethylene-Precursor) Treatments Reveal New Insights into the Physiology of the Ripening Process.

The physiology of Prunus fruit ripening is a complex and not completely understood process. To improve this knowledge, postharvest behavior during the shelf-life period at the transcriptomic level has been studied using high-throughput sequencing analysis (RNA-Seq). Monitoring of fruits has been analyzed after different ethylene regulator treatments, including 1-MCP (ethylene-inhibitor) and Ethrel (ethylene-precursor) in two contrasting selected apricot (Prunus armeniaca L.) and Japanese plum (P. salicina L.) cultivars, 'Goldrich' and 'Santa Rosa'. KEEG and protein-protein interaction network analysis unveiled that the most significant metabolic pathways involved in the ripening process were photosynthesis and plant hormone signal transduction. In addition, previously discovered genes linked to fruit ripening, such as pectinesterase or auxin-responsive protein, have been confirmed as the main genes involved in this process. Genes encoding pectinesterase in the pentose and glucuronate interconversions pathway were the most overexpressed in both species, being upregulated by Ethrel. On the other hand, auxin-responsive protein IAA and aquaporin PIP were both upregulated by 1-MCP in 'Goldrich' and 'Santa Rosa', respectively. Results also showed the upregulation of chitinase and glutaredoxin 3 after Ethrel treatment in 'Goldrich' and 'Santa Rosa', respectively, while photosystem I subunit V psaG (photosynthesis) was upregulated after 1-MCP in both species. Furthermore, the overexpression of genes encoding GDP-L-galactose and ferredoxin in the ascorbate and aldarate metabolism and photosynthesis pathways caused by 1-MCP favored antioxidant activity and therefore slowed down the fruit senescence process.

Mining Candidate Genes Related to Heavy Metals in Mature Melon (Cucumis melo L.) Peel and Pulp Using WGCNA.

The content of metal ions in fruits is inseparable from plant intake of trace elements and health effects in the human body. To understand metal ion content in the fruit and pericarp of melon (Cucumis melo L.) and the candidate genes responsible for controlling this process, we analyzed the metal ion content in distinct parts of melon fruit and pericarp and performed RNA-seq. The results showed that the content of metal ions in melon fruit tissue was significantly higher than that in the pericarp. Based on transcriptome expression profiling, we found that the fruit and pericarp contained elevated levels of DEGs. GO functional annotations included cell surface receptor signaling, signal transduction, organic substance metabolism, carbohydrate derivative binding, and hormone-mediated signaling pathways. KEGG pathways included pectate lyase, pentose and glucuronate interconversions, H+-transporting ATPase, oxidative phosphorylation, plant hormone signal transduction, and MAPK signaling pathways. We also analyzed the expression patterns of genes and transcription factors involved in hormone biosynthesis and signal transduction. Using weighted gene co-expression network analysis (WGCNA), a co-expression network was constructed to identify a specific module that was significantly correlated with the content of metal ions in melon, after which the gene expression in the module was measured. Connectivity and qRT-PCR identified five candidate melon genes, LOC103501427, LOC103501539, LOC103503694, LOC103504124, and LOC107990281, associated with metal ion content. This study provides a theoretical basis for further understanding the molecular mechanism of heavy metal ion content in melon fruit and peel and provides new genetic resources for the study of heavy metal ion content in plant tissues.

Natural Xylooligosaccharides Exert Antitumor Activity via Modulation of Cellular Antioxidant State and TLR4.

It has been recently proven that xylooligosaccharides (XOS) with prebiotic properties have diverse beneficial biological effects including immunomodulatory and antitumor activities. The present article focused on the chemical and biological evaluation of corn-derived commercially available XOS and aimed to elucidate their cytotoxicity and inhibitory potential against tumor cells. Spectrophotometric chemical analyses, Fourier transform infrared spectroscopy, and high-performance liquid chromatography analyses were performed. Antioxidant activity was determined by measuring the oxygen radical absorbance capacity and hydroxyl radical averting capacity. In vitro cytotoxicity assays with human cell lines derived from normal and tumor tissues, assessments of ATP production, mitochondrial membrane potential specific staining, cytokine assays, and molecular docking were used to evaluate the biological activity of XOS. The sample showed significant antioxidant activity, and it was determined that most xylose oligomers in it are composed of six units. XOS exhibited antitumor activity with pronounced inhibitory effect on lysosomes, but mitochondrial functionality was also affected. The production of proinflammatory cytokines by lipopolysaccharide-stimulated U-937 cells was reduced by XOS treatment, which suggested the involvement of Toll-like receptor 4 (TLR4)-mediated signaling in the mechanism of XOS action. Molecular docking analyses confirmed the potential inhibitory interaction between the sample and TLR4. In addition, XOS treatment had significant tumor-cell-specific influence on the glutathione antioxidant system, affecting its balance and thus contributing to the inhibition of cellular viability. The present study elucidated the tumor-inhibitory potential of commercially available XOS that could be utilized in pharmaceutical and food industry providing disease-preventive and therapeutic benefits.

Xylooligosaccharide-mediated gut microbiota enhances gut barrier and modulates gut immunity associated with alterations of biological processes in a pig model.

Xylooligosaccharide (XOS) has tremendous prebiotic potentials for gut health, but the relevant mechanisms are unclear. Herein, we confirmed the positive effects of dietary XOS enhancing gut barrier in a pig model via suppressing the expression of pro-inflammatory cytokines (IL-6 and IL-8). Meanwhile, XOS increased beneficial microbes Lactobacillus and decreased potential pathogenic bacteria. Moreover, XOS augmented microbiota-derived metabolites (mainly butyrate, propionate, and secondary bile acid) to strengthen the gut barrier and regulate gut immunity through activating host G-protein coupled receptors 109a or inhibiting histone deacetylases. Furthermore, XOS attenuated IgA-production and antigen cross-presentation processes. In addition, XOS supplementation led to the alteration of cell proliferation, remodeling of the energy metabolism, activation processes of serial genes or proteins, increased molecular chaperones, and the enhanced ubiquitin-proteasome pathway in cecal cells. Collectively, these results suggest that XOS enhances gut barrier and modulates gut immunity by optimizing gut microbiota and their metabolites, which is associated with alterations of biological processes.

A GH115 α-glucuronidase structure reveals dimerization-mediated substrate binding and a proton wire potentially important for catalysis.

Xylan is a major constituent of plant cell walls and is a potential source of biomaterials, and the derived oligosaccharides have been shown to have prebiotic effects. Xylans can be highly substituted with different sugar moieties, which pose steric hindrance to the xylanases that catalyse the hydrolysis of the xylan backbone. One such substituent is α-D-glucuronic acid, which is linked to the O2' position of the ß-1,4 D-xylopyranoses composing the main chain of xylans. The xylan-specific α-glucuronidases from glycoside hydrolase family 115 (GH115) specifically catalyse the removal of α-D-glucuronic acid (GlcA) or methylated GlcA (MeGlcA). Here, the molecular basis by which the bacterial GH115 member wtsAgu115A interacts with the main chain of xylan and the indirect involvement of divalent ions in the formation of the Michaelis-Menten complex are described. A crystal structure at 2.65 Šresolution of wtsAgu115A originating from a metagenome from an anaerobic digester fed with wastewater treatment sludge was determined in complex with xylohexaose, and Asp303 was identified as the likely general acid. The residue acting as the general base could not be identified. However, a proton wire connecting the active site to the metal site was observed and hence a previous hypothesis suggesting a Grotthuss-like mechanism cannot be rejected. Only a single molecule was found in the asymmetric unit. However, wtsAgu115A forms a dimer with a symmetry-related molecule in the crystal lattice. The xylohexaose moieties of the xylohexaose are recognized by residues from both protomers, thus creating a xylohexaose recognition site at the dimer interface. The dimer was confirmed by analytical size-exclusion chromatography in solution. Kinetic analysis with aldouronic acids resulted in a Hill coefficient of greater than 2, suggesting cooperativity between the two binding sites. Three Ca2+ ions were identified in the wtsAgu115A structures. One Ca2+ ion is of particular interest as it is coordinated by the residues of the loops that also interact with the substrate. Activity studies showed that the presence of Mg2+ or Mn2+ resulted in a higher activity towards aldouronic acids, while the less restrictive coordination geometry of Ca2+ resulted in a decrease in activity.

Ethyl glucuronide in hair detects a high rate of harmful alcohol consumption in presumed non-alcoholic fatty liver disease.

BACKGROUND & AIMS: Non-alcoholic fatty liver disease (NAFLD) and alcohol-related liver disease (ALD) cannot reliably be distinguished by routine diagnostics, and the role of alcohol consumption in metabolic dysfunction-associated fatty liver disease (MAFLD) remains unclear. We investigated alcohol consumption in patients with presumed NAFLD and ALD using novel objective alcohol markers. METHODS: In total, 184 consecutive patients were included in this prospective observational study. Alcohol intake was assessed by ethylglucuronide in hair (hEtG) and urine (uEtG); the utility of these measures for alcohol detection was compared to Alcohol Use Disorders Identification Test-Consumption (AUDIT-C), carbohydrate deficient transferrin (CDT), mean corpuscular volume (MCV), gamma-glutamyltransferase (GGT), and ALD/NAFLD index (ANI). Clinical characteristics of patients with NAFLD and ALD were re-assessed after reclassification based on repeated moderate (≥10 g <60 g EtOH/day) and excessive (≥60 g EtOH/day) alcohol consumption, and patients were retrospectively reclassified based on MAFLD criteria. RESULTS: Repeated moderate to excessive alcohol consumption was detected in 28.6%, 28.5%, and 25.0% of patients with presumed NAFLD, ALD or MAFLD, respectively. ANI score, AUDIT-C, uEtG, and hEtG showed AUCs of 0.628, 0.733, 0.754, and 0.927 for the detection of repeated moderate to excessive alcohol consumption, respectively. The indirect markers CDT, MCV and GGT were not reliable. Patients with repeated moderate or excessive alcohol consumption were significantly more often male, had a significantly lower BMI, and suffered significantly less often from type 2 diabetes or impaired glucose tolerance. CONCLUSIONS: In total, 28.6% of patients with presumed NAFLD, and 25.0% with MAFLD are at risk of alcohol-related liver damage. AUDIT-C, uEtG and hEtG should be used to screen for alcohol consumption in patients with fatty liver disease. LAY SUMMARY: Fatty liver disease can be caused by metabolic factors and/or alcohol consumption. The diagnosis of non-alcoholic fatty liver disease (NAFLD) is based on the exclusion of harmful alcohol consumption, while metabolic dysfunction-associated fatty liver disease (MAFLD), which has been proposed as a new name for NAFLD, is based on the presence of metabolic comorbidities and allows for alcohol consumption. Herein, we show that up to 29% of patients diagnosed with NAFLD and 25% with MAFLD are at risk of alcohol-related liver damage. We show that ethyl glucuronide (a metabolite of alcohol) in the hair and urine can accurately detect potentially harmful alcohol consumption in these patients - as such, these tests should be integrated into routine diagnostic work-up for patients with fatty liver disease.

A Fungal Versatile GH10 Endoxylanase and Its Glycosynthase Variant: Synthesis of Xylooligosaccharides and Glycosides of Bioactive Phenolic Compounds.

The study of endoxylanases as catalysts to valorize hemicellulosic residues and to obtain glycosides with improved properties is a topic of great industrial interest. In this work, a GH10 ß-1,4-endoxylanase (XynSOS), from the ascomycetous fungus Talaromyces amestolkiae, has been heterologously produced in Pichia pastoris, purified, and characterized. rXynSOS is a highly glycosylated monomeric enzyme of 53 kDa that contains a functional CBM1 domain and shows its optimal activity on azurine cross-linked (AZCL)-beechwood xylan at 70 °C and pH 5. Substrate specificity and kinetic studies confirmed its versatility and high affinity for beechwood xylan and wheat arabinoxylan. Moreover, rXynSOS was capable of transglycosylating phenolic compounds, although with low efficiencies. For expanding its synthetic capacity, a glycosynthase variant of rXynSOS was developed by directed mutagenesis, replacing its nucleophile catalytic residue E236 by a glycine (rXynSOS-E236G). This novel glycosynthase was able to synthesize ß-1,4-xylooligosaccharides (XOS) of different lengths (four, six, eight, and ten xylose units), which are known to be emerging prebiotics. rXynSOS-E236G was also much more active than the native enzyme in the glycosylation of a broad range of phenolic compounds with antioxidant properties. The interesting capabilities of rXynSOS and its glycosynthase variant make them promising tools for biotechnological applications.

Understanding the Xylooligosaccharides Utilization Mechanism of Lactobacillus brevis and Bifidobacterium adolescentis: Proteins Involved and Their Conformational Stabilities for Effectual Binding.

Xylooligosaccharides having various degrees of polymerization such as xylobiose, xylotriose, and xylotetraose positively affect human health by interacting with gut proteins. The present study aimed to identify proteins present in gut microflora, such as xylosidase, xylulokinase, etc., with the help of retrieved whole-genome annotations and find out the mechanistic interactions of those with the above substrates. The 3D structures of proteins, namely Endo-1,4-beta-xylanase B (XynB) from Lactobacillus brevis and beta-D-xylosidase (Xyl3) from Bifidobacterium adolescentis, were computationally predicted and validated with the help of various bioinformatics tools. Molecular docking studies identified the effectual binding of these proteins to the xylooligosaccharides, and the stabilities of the best-docked complexes were analyzed by molecular dynamic simulation. The present study demonstrated that XynB and Xyl3 showed better effectual binding toward Xylobiose with the binding energies of - 5.96 kcal/mol and - 4.2 kcal/mol, respectively. The interactions were stabilized by several hydrogen bonding having desolvation energy (- 6.59 and - 7.91). The conformational stabilities of the docked complexes were observed in the four selected complexes of XynB-xylotriose, XynB-xylotetraose, Xyl3-xylobiose, and Xyn3-xylotriose by MD simulations. This study showed that the interactions of these four complexes are stable, which means they have complex metabolic activities among each other. Extending these studies of understanding, the interaction between specific probiotics enzymes and their ligands can explore the detailed design of synbiotics in the future.

Unique features of the bifunctional GH30 from Thermothelomyces thermophila revealed by structural and mutational studies.

Fungal xylanases belonging to family GH30_7, initially categorized as endo-glucuronoxylanases, are now known to differ both in terms of substrate specificity, as well as mode of action. Recently, TtXyn30A, a GH30_7 xylanase from Thermothelomyces thermophila, was shown to possess dual activity, acting on the xylan backbone in both an endo- and an exo- manner. Here, in an effort to identify the structural characteristics that append these functional properties to the enzyme, we present the biochemical characterization of various TtXyn30A mutants as well as its crystal structure, alone, and in complex with the reaction product. An auxiliary catalytic amino acid has been identified, while it is also shown that glucuronic acid recognition is not mediated by a conserved arginine residue, as shown by previously determined GH30 structures.