Glycosyltransferase Research Articles

Characterization of PglJ, a Glycosyltransferase in the Campylobacter concisus N-Linked Protein Glycosylation Pathway that Expands Glycan Diversity.

The Campylobacter genus of Gram-negative bacteria is characterized by the expression of N-linked protein glycosylation (pgl) pathways. As Campylobacter concisus is an emerging human pathogen, a better understanding of the variation of the biosynthetic pathways across the genus is necessary to identify the relationships between protein glycosylation and disease. The pgl pathways of C. concisus strains have been reported to diverge from other Campylobacter in steps after the biosynthesis of N-acetylgalactosamine-α1,3-N,N'-diacetylbacillosamine-α-1-diphosphate undecaprenyl (GalNAc-diNAcBac-PP-Und), which is catalyzed by PglC and PglA, a phosphoglycosyltransferase (PGT) and a glycosyltransferase (GT), respectively. Here we characterize the PglJ GTs from two strains of C. concisus. Chemical synthesis was employed to access the stereochemically defined glycan donor substrates, uridine diphosphate N-acetyl-d-galactosaminuronic acid (UDP-GalNAcA) and uridine diphosphate N-acetyl-d-glucosaminuronic acid (UDP-GlcNAcA), to allow biochemical investigation of PglJ. Evidence for the PglJ substrate specificity structural determinants for the C6″ carboxylate-containing sugar was obtained through variant-based biochemical assays. Additionally, characterization of a UDP-sugar dehydrogenase encoded in the pgl operon, which is similar to the Pseudomonas aeruginosa WbpO responsible for the oxidization of a UDP-HexNAc to UDP-HexNAcA, supports the availability of a UDP-HexNAcA substrate for a GT that incorporates the modified sugar and provides evidence for the presence of a HexNAcA in the N-linked glycan. Utilizing sequence similarity network (SSN) analysis, we identified conserved sequence motifs among PglJ glycosyltransferases, shedding light on substrate preferences and offering predictive insights into enzyme functions across the Campylobacter genus. These studies now allow detailed characterization of the later steps in the pgl pathway in C. concisus strains and provide insights into enzyme substrate specificity determinants for glycan assembly enzymes.

Tree diversity and arbuscular mycorrhizal trees increase soil carbon sequestration and stability in 1-m soils as regulated by microbial CAZymes-coworking in high-latitude Northern Hemisphere forests

Biodiversity loss and tree alternations often occurred in high-latitude Northern Hemisphere forests owing to climatic changes and human disturbance, while their effects on soil carbon sequestration remain unclear. In this paper, tree richness and arbuscular mycorrhizal (AM)-tree dominance experiments were settled in an experimental forest, and soil organic carbon (SOC) and glomalin-related soil protein carbon (Total, TG-C and easily extracted, EEG-C), stability (heterotrophic respiration, Rh, temperature sensitivity, Q10 and humidity sensitivity, Hs), in situ soil CO2, CH4, N2O fluxes, and CAZymes activities from the microbial metagenome technique were measured to identify their impacts and underlying mechanism. Both analysis of variance (ANOVA) and linear regression found tree richness and AM tree loss accompanied by soil carbon sequestration loss of total SOC storage, their mycorrhizal components (TG-C) and Rh. In 0–20 cm soils, high tree richness quadrats had 16–126.6 % higher SOC, TG-C, Rh, and in situ N2O flux, while AM-dominated stands had 34–39 % higher SOC and Rh than the EcM-dominated stands. In the 1 m soil profile, the effect size from tree richness and AM trees were more evident than the 0–20 cm soils. Compared with the richness gradient, much significant differences occurred between AM- and EcM-dominated stands. Structural equation modeling found that tree richness directly improved C stability, while the SOC accrual from the tree richness enrichment and AM-tree dominance were indirectly achieved by their decreasing in multiple CAZymes activities. Of CAZymes, glycoside hydrolases, polysaccharide lyases, carbohydrate esterases, glycosyl transferases, auxiliary activities, and carbohydrate-binding modules cooperated in their regulation of the soil C accrual. Our findings support carbon sequestration via biodiversity conservation and proper forest management related to tree mycorrhizal symbionts.

Glycosyltransferase 8 domain-containing protein 1 (GLT8D1) is a UDP-dependent galactosyltransferase

Glycosyltransferases (GTs) are enzymes that catalyze the formation of glycosidic bonds and hundreds of GTs have been identified so far in humans. Glycosyltransferase 8 domain-containing protein 1 (GLT8D1) has been associated with central nervous system diseases and cancer. However, evidence on its enzymatic properties, including its substrates, has been scarcely described. In this paper, we have produced and purified recombinant secretory GLT8D1. The enzyme was found to be N-glycosylated. Differential scanning fluorimetry was employed to analyze the stabilization of GLT8D1 by Mn2+ and nucleotides, revealing UDP as the most stabilizing nucleotide scaffold. GLT8D1 displayed glycosyltransferase activity from UDP-galactose onto N-acetylgalactosamine but with a low efficiency. Modeling of the structure revealed similarities with other GT-A fold enzymes in CAZy family GT8 and glycosyltransferases in other families with galactosyl-, glucosyl-, and xylosyltransferase activities, each with retaining catalytic mechanisms. Our study provides novel structural and functional insights into the properties of GLT8D1 with implications in pathological processes.

Gut microbiota changes in Hexagrammos otakii during development and association with diets based on metagenomics sequencing

The development of microbial communities is important for the growth of the fat greenling Hexagrammos otakii; however, the relationship between microbial communities and fish diet is poorly understood. Here, we used metagenomics sequencing to investigate the gut microbiota of H. otakii at three key time points during development. The gut microbial diversity and community composition of H. otakii presented stage-specific signatures and appeared to shift with different diets and environmental conditions such as NH4 + -NO2- etc. Metagenomics analysis indicated that the dominant phyla were Proteobacteria and Firmicutes. With dietary changes from rotifers and Artemia to formulated feed, the dominant genera shifted from Vibrio and Photobacterium to Lactococcus, and variations in the gut microbiota increased with age. Based on metagenomics functional annotation using the Kyoto Encyclopedia of Genes and Genomes database, growth and diet at different stages affected the metabolic function of the gut microbiome by activating carbohydrate and amino acid metabolism while weakening lipid and nucleotide metabolism. In addition, as predicted by the Carbohydrate-Active enZymes database, glycoside hydrolase and glycosyl transferases may be involved in food degradation. Our results suggest that the H. otakii gut microbiota assembly was coupled to animal development and host age-associated biotic features and changes. Diet had a considerable effect on these distinct communities at different times, indicating that the fish gut microbiota rapidly adapted to dietary shifts. The microbiota therefore likely plays an indispensable role in nutrient turnover and fermentation. These findings can provide guidance for the development of probiotics and the application of feed additives.

Carbohydrate-active enzyme profiles of Lactiplantibacillus plantarum strain 84-3 contribute to flavor formation in fermented dairy and vegetable products

Microbes are critical for flavor formation in fermented foods; however, their mechanisms of action are not fully understood. The microbial composition of 51 dairy and 47 vegetable products was functionally annotated and the carbohydrate-active enzyme (CAZyme) profiles of Lactiplantibacillus plantarum 84-3 (Lp84-3), isolated from dairy samples, can promote resistant starch (RS) degradation, were analyzed. Lactobacillus, Streptococcus, and Lactococcus were the predominant genera in dairy products, whereas the major genera in vegetables were Lactobacillus, Weissella, and Carnimonas. Phages from Siphoviridae, Myoviridae, and Herelleviridae were also present in dairy products. Additionally, the glycosyl hydrolase (GHs) family members GH1 and GH13 and the glycosyltransferase (GTs) family members GT2 and GT4 were abundant in Lp84-3. Moreover, Lp84-3 was enriched in butanoate metabolism enzymes and butanoate metabolite compounds. Therefore, fermented food microbes, especially Lp84-3, have an abundant repertoire of enzymes that promote flavor production, as starter improving the flavor of fermented dairy and vegetable products.

Genome-wide adenine N6-methylation map reveals epigenomic regulation of lipid accumulation in Nannochloropsis

Epigenetic marks on histones and DNA, such as DNA methylation at N6-adenine (6mA), play crucial roles in gene expression and genome maintenance, but their deposition and function in microalgae remain largely uncharacterized. Here, we report a genome-wide 6mA map for the model industrial oleaginous microalga Nannochloropsis oceanica produced by single-molecule real-time sequencing. Found in 0.1% of adenines, 6mA sites are mostly enriched at the AGGYV motif, more abundant in transposons and 3′ untranslated regions, and associated with active transcription. Moreover, 6mA gradually increases in abundance along the direction of gene transcription and shows special positional enrichment near splicing donor and transcription termination sites. Highly expressed genes tend to show greater 6mA abundance in the gene body than do poorly expressed genes, indicating a positive interaction between 6mA and general transcription factors. Furthermore, knockout of the putative 6mA methylase NO08G00280 by genome editing leads to changes in methylation patterns that are correlated with changes in the expression of molybdenum cofactor, sulfate transporter, glycosyl transferase, and lipase genes that underlie reductions in biomass and oil productivity. By contrast, knockout of the candidate demethylase NO06G02500 results in increased 6mA levels and reduced growth. Unraveling the epigenomic players and their roles in biomass productivity and lipid metabolism lays a foundation for epigenetic engineering of industrial microalgae.

Differences in microbial communities and potato growth in two soil types under organic cultivation.

Organic agriculture plays a positive role in promoting genetic diversity, including living organisms, plants, and cultivated crops in the soil. However, few comparative studies reported whether different soil types were suitable for organic cultivation. In this study, loam and clay-loam soils under continuous organic cultivation were analyzed. The results showed that there were no significant differences between two soil types in soil pH, bulk density, total porosity, moisture content and three soil phases. The capillary porosity and organic matter content of loam were significantly higher than those of clay-loam. Compared with clay-loam soil, the contents of total nitrogen, phosphorus, potassium, calcium, zinc and silicon in loam soil were also significantly higher. The microbial diversity was higher in loam and the dominant microbes differed between the two soils. Glycosyl transferases and carbohydrate esterases were enriched in loam, whereas glycoside hydrolases and carbohydrate-binding modules were enriched in clay loam. The potato yield in loam was significantly higher than that in clay loam. Among the tuber quality indicators, the protein content of potatoes in loam was higher than that in clay-loam, but the reducing sugar content was lower for loam than for clay-loam. In conclusion, compared with clay loam, loam was more suitable for organic cultivation of potatoes on account of the high contents of nitrogen, phosphorus, and potassium and the rich microbial community, thus promoting a high yield of tubers. This study provided a theoretical reference for the selection of soil type suitable for organic cultivation.

Analysis of Aureobasidium pullulans LB83 secretome reveals distinct carbohydrate active enzymes for biomass saccharification

Aureobasidium pullulans LB83 is a versatile biocatalyst that produces a plethora of bioactive products thriving on a variety of feedstocks under the varying culture conditions. In our last study using this microorganism, we found cellulase activity (FPase, 2.27 U/ml; CMCase, 7.42 U/ml) and other plant cell wall degrading enzyme activities grown on sugarcane bagasse and soybean meal as carbon source and nitrogen, respectively. In the present study, we provide insights on the secretome analysis of this enzymatic cocktail. The secretome analysis of A. pullulans LB83 by Liquid Chromatography coupled to Mass Spectroscopy (LC-MS/MS) revealed 38 classes of Carbohydrate Active enZymes (CAZymes) of a total of 464 identified proteins. These CAZymes consisted of 21 glycoside hydrolases (55.26%), 12 glycoside hydrolases harboring carbohydrate-binding module (31.58%), 4 carbohydrate esterases (10.53%) and one glycosyl transferase (2.63%). To the best of our knowledge, this is the first report on the secretome analysis of A. pullulans LB83.

PSI-13 Effects of a Postbiotic Program Consisting of Liquid and Solid Saccharomyces Cerevisiae Fermentation Products on Ruminal Microbiome Composition and Microbial Functions of Finishing Beef Steers

Abstract We assessed the effects of a postbiotic program consisting of liquid and solid Saccharomyces cerevisiae fermentation products (SCFP) on the ruminal microbiome and fermentation characteristics of beef steers. Eight Holstein steers (BW 467 ± 13.9 kg) with rumen cannulas were used in a crossover design study with 21-d per period and 7-d washout in between periods. Steers were blocked based on initial BW and assigned to one of two treatments: 1) Control, basal diet, or 2) SCFP, basal diet plus one day feeding of LiquiCare RTU (Diamond V, Cedar Rapids, IA; dosed via rumen cannula at 11 mL/100 kg BW), followed by daily feeding of 12 g/d NaturSafe (Diamond V, top-dressed). Rumen fluid samples were collected on d 0, 1, 2, 3, 5 and 21 at 0, 4, 8, or 12 h post morning feeding via rumen cannula for pH, VFA and NH3-N analyses. Samples collected at 4 h post morning feeding on d 0, 2 and 21 were subjected to DNA extraction and shotgun Nanopore sequencing to determine microbiome composition and carbohydrate active enzymes (CAZy) functional potential. Statistical model included the fixed effects of treatment, period, day and their interactions and random effect of animal. Feeding SCFP tended to reduce ruminal lactate (0.09 vs. 0.02 mM, P = 0.10), and reduced ruminal NH3-N (4.86 vs. 6.83 mg/dL, P = 0.04) on d 21. Ruminal total VFA concentration was increased (P = 0.01) by feeding SCFP 4-h post-feeding on d 5 and numerically increased 4-h post-feeding on day 21 (133.3 vs. 151.2 mM, P = 0.12). Treatments did not affect species or CAZy alpha- and beta-diversity. Steers received liquid postbiotic on d 2 exhibited a positive correlation (P < 0.05) between propionate molar proportion and glycosyl transferases (GT), glycoside hydrolases (GH), carbohydrate esterases, and carbohydrate binding modules (CBM); and a negative correlation (P < 0.05) between acetate molar proportion and polysaccharide lyases, GT, GH, and CBM, which was not observed in Control. Additionally, SCFP steers had higher (P = 0.05) CBM functional potential on d 21 than on d 0, while CAZy class abundances did not change over time in Control steers. Differential network analysis indicated a greater normalized degree for GT in the SCFP steers compared with Control on d 21. There were 2, 6 and 12 differently abundant species (P < 0.01) between SCFP and Control on d 0, 2 and 21, respectively, suggesting supplementing SCFP altered the abundances of more species over time. Our results showed that the postbiotic program altered the metabolic pathways of ruminal microbes toward greater propionate production, and improved abundance of carbohydrate binding modules. Our results also showed that glycosyl transferases had a greater promotive role in the ruminal enzymatic network of SCFP steers.

Exploring 3-O-glycosylations of 20(R)-dammarane ginsenosides and the catalytic mechanism underlying the stereoselectivity with the combined assistance of AlphaFold 2 and molecular docking

Glycosylation at C3-OH is the favorable modification for pharmaceutical activities and diversity expansion of 20(R)-dammarane ginsenosides. The 3-O-glycosylation, exclusively occurring in 20(R)-PPD ginsenosides, has never been achieved in 20(R)-PPT ginsenosides. Herein, 3-O-glycosylation of 20(R)-PPT enabled by a glycosyltransferase (GT) OsSGT2 was achieved with the combined assistance of AlphaFold 2 and molecular docking. Firstly, we combined AlphaFold2 algorithm and molecular docking to predict interactions between 20(R)-PPT and candidate GTs. A catalytically favorable binding geometry was thus identified in the OsSGT2–20(R)-PPT complex, suggesting OsSGT2 might act on 20(R)-PPT. The enzymatic assays demonstrated that OsSGT2 reacted with varied sugar donors to form 20(R)-PPT 3-O-glycosides, exhibiting donor promiscuity. Additionally, OsSGT2 displayed acceptor promiscuity, catalyzing 3-O-glucosylation of 20(R/S)-PPT, 20(R/S)-PPD and 20(R/S)-Rh1, respectively. Protein engineering on OsSGT2 was thus performed to probe its catalytic mechanism underlying its stereoselectivity. The W207A mutant preferred 20(S)-dammarane aglycons, while F395Q/A396G(QG) displayed a conversion enhancement towards both 20(R/S)-dammarane aglycons. The QG mutant was then used to synthesize 20(R)-PPT 3-O-glucoside, which displayed a moderate angiotensin-converting enzyme inhibitory effect with an IC50 of 27.5 ± 4.7 μM, superior to that of its 20(S)-epimer, with the combined assistance of target fishing and reverse docking. The water solubility of 20(R)-PPT 3-O-glucoside increased as well.

Genome-wide identification reveals conserved carbohydrate-active enzyme repertoire in termites

Termites play an important role as decomposers of organic matter in forests by utilizing their gut symbionts and associated carbohydrate-active enzymes (CAZymes) to digest wood materials. However, there is a limited understanding of the entire repertoire of CAZymes and their evolution in termite genomes. Here we identified the gene families of CAZymes in publicly available termite genomes and analyzed the evolution of abundant gene families. We found that 79 CAZyme gene families from the carbohydrate-binding module and four CAZyme classes, including glycosyl transferase (GT), glycoside hydrolase (GH), auxiliary activity (AA) and carbohydrate esterase (CE), were present in termites with minor variations across termite species except for a few gene families. The gene trees of the large and conserved gene families have several groups of genes from all species, and each group encodes enzymes with complete corresponding domains. Three gene families, namely GT1, GH1 and AA3, exhibited significant variations in gene numbers and experienced several losses and a few duplications, which might be related to their rich gut symbionts and newly gained functions. Furthermore, the overall expression of CAZymes appears to have a caste- and tissue-specific pattern, reflecting a division of labor in termite colonies. Overall, these results reveal a likely stable CAZyme repertoire in termites and pave the way for further research on the functional contribution of termites to wood digestion.

Mechanism and evolutionary analysis of Yarrowia lipolytica CA20 capable of producing erythritol with a high yield based on comparative genomics.

Combined mutagenesis is widely applied for the breeding of robust Yarrowia lipolytica used in the production of erythritol. However, the changes of genome after mutagenesis remains unclear. This study aimed to unravel the mechanism involved in the improved erythritol synthesis of CA20 and the evolutionary relationship between different Y. lipolytica by comparative genomics analysis. The results showed that the genome size of Y. lipolytica CA20 was 20,420,510 bp, with a GC content of 48.97%. There were 6330 CDS and 649 ncRNA (non-coding RNA) in CA20 genome. Average nucleotide identity (ANI) analysis showed that CA20 genome possessed high similarity (ANI > 99.50%) with other Y. lipolytica strains, while phylogenetic analysis displayed that CA20 was classified together with Y. lipolytica IBT 446 and Y. lipolytica H222. CA20 shared 5342 core orthologous genes with the 8 strains while harbored 65 specific genes that mainly participated in the substrate and protein transport processes. CA20 contained 166 genes coding for carbohydrate-active enzymes (CAZymes), which was more than that found in other strains (108－137). Notably, 4, 2, and 13 different enzymes belonging to glycoside hydrolases (GHs), glycosyltransferases (GTs), and carbohydrate esterases (CEs), respectively, were only found in CA20. The enzymes involved in the metabolic pathway of erythritol were highly conserved in Y. lipolytica, except for transaldolase (TAL1). In addition, the titer and productivity of erythritol by CA20 were 190.97 g/L and 1.33 g/L/h, respectively, which were significantly higher than that of WT5 wherein 128.61 g/L and 0.92 g/L/h were obtained (P< 0.001). Five frameshift mutation genes and 15 genes harboring nonsynonymous mutation were found in CA20 compared with that of WT5. Most of these genes were involved in the cell division, cell wall synthesis, protein synthesis, and protein homeostasis maintenance. These findings suggested that the genome of Y. lipolytica is conserved during evolution, and the variance of living environment is one important factor leading to genome divergence. The varied number of CAZymes existed in Y. lipolytica is one factor that contributes to the performance difference. The increased synthesis of erythritol by Y. lipolytica CA20 is correlated with the improvement of the stability of cell structure and internal environment. The results of this study provide a basis for the directional breeding of robust strains used in erythritol production.

A Legionella toxin exhibits tRNA mimicry and glycosyl transferase activity to target the translation machinery and trigger a ribotoxic stress response.

A widespread strategy employed by pathogens to establish infection is to inhibit host-cell protein synthesis. Legionella pneumophila, an intracellular bacterial pathogen and the causative organism of Legionnaires' disease, secretes a subset of protein effectors into host cells that inhibit translation elongation. Mechanistic insights into how the bacterium targets translation elongation remain poorly defined. We report here that the Legionella effector SidI functions in an unprecedented way as a transfer-RNA mimic that directly binds to and glycosylates the ribosome. The 3.1 Å cryo-electron microscopy structure of SidI reveals an N-terminal domain with an 'inverted L' shape and surface-charge distribution characteristic of tRNA mimicry, and a C-terminal domain that adopts a glycosyl transferase fold that licenses SidI to utilize GDP-mannose as a sugar precursor. This coupling of tRNA mimicry and enzymatic action endows SidI with the ability to block protein synthesis with a potency comparable to ricin, one of the most powerful toxins known. In Legionella-infected cells, the translational pausing activated by SidI elicits a stress response signature mimicking the ribotoxic stress response, which is activated by elongation inhibitors that induce ribosome collisions. SidI-mediated effects on the ribosome activate the stress kinases ZAKα and p38, which in turn drive an accumulation of the protein activating transcription factor 3 (ATF3). Intriguingly, ATF3 escapes the translation block imposed by SidI, translocates to the nucleus and orchestrates the transcription of stress-inducible genes that promote cell death, revealing a major role for ATF3 in the response to collided ribosome stress. Together, our findings elucidate a novel mechanism by which a pathogenic bacterium employs tRNA mimicry to hijack a ribosome-to-nuclear signalling pathway that regulates cell fate.

Biochemical data documenting variations in mucilage polysaccharides in a range of glycosyltransferase mutants

During Arabidopsis seed coat development, copious amounts of mucilage polysaccharides are produced in the epidermal cells. When hydrated on imbibition, these polysaccharides expand and are released to encapsulate the seed as a two-layered hydrogel. Polysaccharides are synthesized from UDP-sugars by glycosyltransferases (GTs) and several GTs, with differing activities, have been identified that contribute to mucilage polysaccharide synthesis. How these GTs orchestrate production of the complex polysaccharides found in mucilage remains to be determined. In this study, we generated a range of multiple GT mutants using either CRISPR/Cas9 targeted mutation or genetic crosses of existing T-DNA insertion mutants. Four traits for mucilage amounts or macromolecular properties were examined for four replicate seed lots from 31 different GT mutant combinations. This data provides a valuable resource for future genetic, biochemical, structural, and functional studies of the roles and properties of polysaccharides present in Arabidopsis mucilage and the relative contributions of different GTs to mucilage production.

Erratum: Mutation of putative glycosyl transferases PslC and PslI confers susceptibility to antibiotics and leads to drastic reduction in biofilm formation in Pseudomonas aeruginosa.

Erratum: Mutation of putative glycosyl transferases PslC and PslI confers susceptibility to antibiotics and leads to drastic reduction in biofilm formation in Pseudomonas aeruginosa.

Catalytic Mechanism of Nonglycosidic C-N Bond Formation by the Pseudoglycosyltransferase Enzyme VldE.

The pseudoglycosyltransferase (PsGT) enzyme VldE is a homologue of the retaining glycosyltransferase (GT) trehalose 6-phosphate synthase (OtsA) that catalyzes a coupling reaction between two pseudo-sugar units, GDP-valienol and validamine 7-phosphate, to give a product with α,α-N-pseudo-glycosidic linkage. Despite its biological importance and unique catalytic function, the molecular bases for its substrate specificity and reaction mechanism are still obscure. Here, we report a comparative mechanistic study of VldE and OtsA using various engineered chimeric proteins and point mutants of the enzymes, X-ray crystallography, docking studies, and kinetic isotope effects. We found that the distinct substrate specificities between VldE and OtsA are most likely due to topological differences within the hot spot amino acid regions of their N-terminal domains. We also found that the Asp158 and His182 residues, which are in the active site, play a significant role in the PsGT function of VldE. They do not seem to be directly involved in the catalysis but may be important for substrate recognition or contribute to the overall architecture of the active site pocket. Moreover, results of the kinetic isotope effect experiments suggest that VldE catalyzes a C-N bond formation between GDP-valienol and validamine 7-phosphate via an SNi-like mechanism. The study provides new insights into the substrate specificity and catalytic mechanism of a member of the growing family of PsGT enzymes, which may be used as a basis for developing new PsGTs from GTs.

Selective turn-on sensing of adenosine diphosphate and phosphate anions by ruthenium (II) polypyridine anchored p-tert-butylcalix[4]arene platform

BackgroundNucleoside polyphosphate (NPP) anions are important for enzymatic activity and should be monitored by scientists in industry and medicine. By elucidating enzyme kinetics and processes, it aids in the discovery of effective inhibitors and activators. Nucleoside polyphosphate (NPP) anions are used by kinases, GTPases, and glycosyltransferases (GTs). Phosphorylation of certain amino acid residues (Ser, Thr, and Tyr) on proteins requires the breakdown of ATP by protein kinases, which produces ADP. Protein kinases, breakdown of ATP, and NPP are the focus of oncology drug development because the aberrant control of kinase activity is a common cause of cancer. ResultsHowever, a discriminative turn-on fluorescent property is exhibited by non-fluorescent p-tertbutylcalix[4]arene modified 1,2,3-triazole containing bis-ruthenium polypyridyl complex (RL) upon the addition of phosphate anions such as (dihydrogen pyrophosphate (H2P2O72−) and dihydrogen phosphate (H2PO4−)) in CH3CN solvent and Adenosine Diphosphate (ADP) in CH3CN/HEPES (pH = 7.4) buffer (9/1, v/v). The probe RL shows a better-recognizing ability with pyrophosphate anion (H2P2O72−) than dihydrogen phosphate anion (H2PO4−). With H2P2O72− and H2PO4− anions, the RL detection limit was calculated to be as low as 83 nM and 198 nM, respectively. SignificanceThe calix[4]arene macrocycle's excellent size and binding cone conformation make it a good host–guest interface for the pyrophosphate anion and ADP. The bis-ruthenium polypyridyl complex's connection to the p-tertbutyl calix[4]arene moiety creates the ADP selectivity turn-on sensor. When moving from mono-nuclear to bi-nuclear ruthenium complex anchored on p-tertbutyl calix[4]arene, the probe can differentiate ADP, ATP, and AMP. Furthermore, this platform is a great resource for creating devices to simultaneously assess phosphate anions in environmental samples.

Mutation of putative glycosyl transferases PslC and PslI confers susceptibility to antibiotics and leads to drastic reduction in biofilm formation in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic, multidrug-resistant pathogen capable of adapting to numerous environmental conditions and causing fatal infections in immunocompromised patients. The predominant lifestyle of P. aeruginosa is in the form of biofilms, which are structured communities of bacteria encapsulated in a matrix containing exopolysaccharides, extracellular DNA (eDNA) and proteins. The matrix is impervious to antibiotics, rendering the bacteria tolerant to antimicrobials. P. aeruginosa also produces a plethora of virulence factors such as pyocyanin, rhamnolipids and lipopolysaccharides among others. In this study we present the molecular characterization of pslC and pslI genes, of the exopolysaccharide operon, that code for putative glycosyltransferases. PslC is a 303 amino acid containing putative GT2 glycosyltrasferase, whereas PslI is a 367 aa long protein, possibly functioning as a GT4 glycosyltransferase. Mutation in either of these two genes results in a significant reduction in biofilm biomass with concomitant decline in c-di-GMP levels in the bacterial cells. Moreover, mutation in pslC and pslI dramatically increased susceptibility of P. aeruginosa to tobramycin, colistin and ciprofloxacin. Additionally, these mutations also resulted in an increase in rhamnolipids and pyocyanin formation. We demonstrate that elevated rhamnolipids promote a swarming phenotype in the mutant strains. Together these results highlight the importance of PslC and PslI in the biogenesis of biofilms and their potential as targets for increased antibiotic susceptibility and biofilm inhibition.

Fungal Glycosidases in Sporothrix Species and Candida albicans.

Glycoside hydrolases (GHs) are enzymes that participate in many biological processes of fungi and other organisms by hydrolyzing glycosidic linkages in glycosides. They play fundamental roles in the degradation of carbohydrates and the assembly of glycoproteins and are important subjects of studies in molecular biology and biochemistry. Based on amino acid sequence similarities and 3-dimensional structures in the carbohydrate-active enzyme (CAZy), they have been classified in 171 families. Members of some of these families also exhibit the activity of trans-glycosydase or glycosyl transferase (GT), i.e., they create a new glycosidic bond in a substrate instead of breaking it. Fungal glycosidases are important for virulence by aiding tissue adhesion and colonization, nutrition, immune evasion, biofilm formation, toxin release, and antibiotic resistance. Here, we review fungal glycosidases with a particular emphasis on Sporothrix species and C. albicans, two well-recognized human pathogens. Covered issues include a brief account of Sporothrix, sporotrichosis, the different types of glycosidases, their substrates, and mechanism of action, recent advances in their identification and characterization, their potential biotechnological applications, and the limitations and challenges of their study given the rather poor available information.

Rewiring the pneumococcal capsule pathway for investigating glycosyltransferase specificity and genetic glycoengineering.

Virtually all living cells are covered with glycans. Their structures are primarily controlled by the specificities of glycosyltransferases (GTs). GTs typically adopt one of the three folds, namely, GT-A, GT-B, and GT-C. However, what defines their specificities remain poorly understood. Here, we developed a genetic glycoengineering platform by reprogramming the capsular polysaccharide pathways in Streptococcus pneumoniae to interrogate GT specificity and manipulate glycan structures. Our findings suggest that the central cleft of GT-B enzymes is important for determining acceptor specificity. The constraint of the glycoengineering platform was partially alleviated when the specificity of the precursor transporter was reduced, indicating that the transporter contributes to the overall fidelity of glycan synthesis. We also modified the pneumococcal capsule to produce several medically important mammalian glycans, as well as demonstrated the importance of regiochemistry in a glycosidic linkage on binding lung epithelial cells. Our work provided mechanistic insights into GT specificity and an approach for investigating glycan functions.