Biosynthesis and Transport of Nucleotide Sugars for Plant Hemicellulose

Hemicellulose is entangled with cellulose through hydrogen bonds and meanwhile acts as a bridge for the deposition of lignin monomer in the secondary wall. Therefore, hemicellulose plays a vital role in the utilization of cell wall biomass. Many advances in hemicellulose research have recently been made, and a large number of genes and their functions have been identified and verified. However, due to the diversity and complexity of hemicellulose, the biosynthesis and regulatory mechanisms are yet unknown. In this review, we summarized the types of plant hemicellulose, hemicellulose-specific nucleotide sugar substrates, key transporters, and biosynthesis pathways. This review will contribute to a better understanding of substrate-level regulation of hemicellulose synthesis.

Metabolic heritage mapping: heterogenous pools of cytoplasmic nucleotide sugars are selectively utilized by various glycosyltransferases

Biosynthesis of macromolecules requires precursors such as sugars or amino acids, originating from exogenous/dietary sources, reutilization/salvage of degraded molecules or de novo synthesis. Since these sources are assumed to contribute to one homogenous pool, their individual contributions are often overlooked. Protein glycosylation uses monosaccharides from all the above sources to produce nucleotide sugars required to assemble hundreds of distinct glycans. Here we demonstrate that cells identify the origin/heritage of the monosaccharide, fucose, for glycosylation. We measured the contribution of GDP-fucose from each of these sources for glycan synthesis and found that different fucosyltransferases, individual glycoproteins, and linkage-specific fucose residues identify and select different GDP-fucose pools dependent on their heritage. This supports the hypothesis that GDP-fucose exists in multiple, distinct pools, not as a single homogenous pool. The selection is tightly regulated since the overall pool size remains constant. We present novel perspectives on monosaccharide metabolism, which may have general applicability.

Metabolic Engineering of Corynebacterium glutamicum for Production of UDP-N-Acetylglucosamine

Uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) is an acetylated amino sugar nucleotide that naturally serves as precursor in bacterial cell wall synthesis and is involved in prokaryotic and eukaryotic glycosylation reactions. UDP-GlcNAc finds application in various fields including the production of oligosaccharides and glycoproteins with therapeutic benefits. At present, nucleotide sugars are produced either chemically or in vitro by enzyme cascades. However, chemical synthesis is complex and non-economical, and in vitro synthesis requires costly substrates and often purified enzymes. A promising alternative is the microbial production of nucleotide sugars from cheap substrates. In this study, we aimed to engineer the non-pathogenic, Gram-positive soil bacterium Corynebacterium glutamicum as a host for UDP-GlcNAc production. The native glmS, glmU, and glmM genes and glmM of Escherichia coli, encoding the enzymes for UDP-GlcNAc synthesis from fructose-6-phosphate, were over-expressed in different combinations and from different plasmids in C. glutamicum GRS43, which lacks the glucosamine-6-phosphate deaminase gene (nagB) for glucosamine degradation. Over-expression of glmS, glmU and glmM, encoding glucosamine-6-phosphate synthase, the bifunctional glucosamine-1-phosphate acetyltransferase/N-acetyl glucosamine-1-phosphate uridyltransferase and phosphoglucosamine mutase, respectively, was confirmed using activity assays or immunoblot analysis. While the reference strain C. glutamicum GlcNCg1 with an empty plasmid in the exponential growth phase contained intracellularly only about 0.25 mM UDP-GlcNAc, the best engineered strain GlcNCg4 accumulated about 14 mM UDP-GlcNAc. The extracellular UDP-GlcNAc concentrations in the exponential growth phase did not exceed 2 mg/L. In the stationary phase, about 60 mg UDP-GlcNAc/L was observed extracellularly with strain GlcNCg4, indicating the potential of C. glutamicum to produce and to release the activated sugar into the culture medium. To our knowledge, the observed UDP-GlcNAc levels are the highest obtained with microbial hosts, emphasizing the potential of C. glutamicum as a suitable platform for activated sugar production.

Single nucleotide polymorphisms within the cps loci: another potential source of clinically important genetic variation for Streptococcus pneumoniae ?

The Streptococcus pneumoniae capsule is essential for disease pathogenesis, suggesting that even minor genetic changes within the cps locus could potentially have important consequences. Arends et al. have identified 79 different non-synonymous SNPs in the cps locus of 338 19A serotype strains, and shown significant variations between strains in nucleotide sugars content and capsule shedding. Further work is required to characterise whether any of these changes have important functional consequences on capsule/host interactions.

Cell surface carbohydrates of symbiotic dinoflagellates and their role in the establishment of cnidarian–dinoflagellate symbiosis

Symbiodiniaceae algae are often photosymbionts of reef-building corals. The establishment of their symbiosis resembles a microbial infection where eukaryotic pattern recognition receptors (e.g. lectins) are thought to recognize a specific range of taxon-specific microbial-associated molecular patterns (e.g. glycans). The present study used the sea anemone, Exaiptasia diaphana and three species of Symbiodiniaceae (the homologous Breviolum minutum, the heterologous-compatible Cladocopium goreaui and the heterologous-incompatible Fugacium kawagutii) to compare the surface glycomes of three symbionts and explore the role of glycan–lectin interactions in host–symbiont recognition and establishment of symbiosis. We identified the nucleotide sugars of the algal cells, then examined glycans on the cell wall of the three symbiont species with monosaccharide analysis, lectin array technology and fluorescence microscopy of the algal cell decorated with fluorescently tagged lectins. Armed with this inventory of possible glycan moieties, we then assayed the ability of the three Symbiodiniaceae to colonize aposymbiotic E. diaphana after modifying the surface of one of the two partners. The Symbiodiniaceae cell-surface glycome varies among algal species. Trypsin treatment of the alga changed the rate of B. minutum and C. goreaui uptake, suggesting that a protein-based moiety is an essential part of compatible symbiont recognition. Our data strongly support the importance of D-galactose (in particular β-D-galactose) residues in the establishment of the cnidarian–dinoflagellate symbiosis, and we propose a potential involvement of L-fucose, D-xylose and D-galacturonic acid in the early steps of this mutualism.

Genetically encoded green fluorescent biosensors for monitoring UDP-GlcNAc in live cells

Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) is a nucleotide sugar used by glycosyltransferases to synthesize glycoproteins, glycosaminoglycans, glycolipids, and glycoRNA. UDP-GlcNAc also serves as the donor substrate for the formation of O-GlcNAc, a dynamic intracellular protein modification involved in diverse signaling and disease processes. UDP-GlcNAc is thus a central metabolite connecting nutrition, metabolism, signaling, and disease. There is a great interest in monitoring UDP-GlcNAc in biological systems. Here, we present the first genetically encoded, green fluorescent UDP-GlcNAc sensor (UGAcS), an optimized insertion of a circularly permuted green fluorescent protein (cpGFP) into an inactive mutant of an E. coli UDP-GlcNAc transferase, for ratiometric monitoring of UDP-GlcNAc dynamics in live mammalian cells. Although UGAcS responds to UDP-GlcNAc quite selectively among various nucleotide sugars, UDP and UTP interfere with the response. We thus developed another biosensor named UXPS, which is responsive to UDP and UTP but not UDP-GlcNAc. We demonstrated the use of the biosensors to follow UDP-GlcNAc levels in cultured mammalian cells perturbed with nutritional changes, pharmacological inhibition, and knockdown or overexpression of key enzymes in the UDP-GlcNAc synthesis pathway. We further utilized the biosensors to monitor UDP-GlcNAc concentrations in pancreatic MIN6 β-cells under various culture conditions.

Two RmlC homologs catalyze dTDP-4-keto-6-deoxy-d-glucose epimerization in Pseudomonas putida KT2440

Abstractl-Rhamnose is an important monosaccharide both as nutrient source and as building block in prokaryotic glycoproteins and glycolipids. Generation of those composite molecules requires activated precursors being provided e. g. in form of nucleotide sugars such as dTDP-β-l-rhamnose (dTDP-l-Rha). dTDP-l-Rha is synthesized in a conserved 4-step reaction which is canonically catalyzed by the enzymes RmlABCD. An intact pathway is especially important for the fitness of pseudomonads, as dTDP-l-Rha is essential for the activation of the polyproline specific translation elongation factor EF-P in these bacteria. Within the scope of this study, we investigated the dTDP-l-Rha-biosynthesis route of Pseudomonas putida KT2440 with a focus on the last two steps. Bioinformatic analysis in combination with a screening approach revealed that epimerization of dTDP-4-keto-6-deoxy-d-glucose to dTDP-4-keto-6-deoxy-l-mannose is catalyzed by the two paralogous proteins PP_1782 (RmlC1) and PP_0265 (RmlC2), whereas the reduction to the final product is solely mediated by PP_1784 (RmlD). Thus, we also exclude the distinct RmlD homolog PP_0500 and the genetically linked nucleoside diphosphate-sugar epimerase PP_0501 to be involved in dTDP-l-Rha formation, other than suggested by certain databases. Together our analysis contributes to the molecular understanding how this important nucleotide-sugar is synthesized in pseudomonads.

β-Glucan phosphorylases in carbohydrate synthesis

β-Glucan phosphorylases are carbohydrate-active enzymes that catalyze the reversible degradation of β-linked glucose polymers, with outstanding potential for the biocatalytic bottom-up synthesis of β-glucans as major bioactive compounds. Their preference for sugar phosphates (rather than nucleotide sugars) as donor substrates further underlines their significance for the carbohydrate industry. Presently, they are classified in the glycoside hydrolase families 94, 149, and 161 (www.cazy.org). Since the discovery of β-1,3-oligoglucan phosphorylase in 1963, several other specificities have been reported that differ in linkage type and/or degree of polymerization. Here, we present an overview of the progress that has been made in our understanding of β-glucan and associated β-glucobiose phosphorylases, with a special focus on their application in the synthesis of carbohydrates and related molecules. Key points • Discovery, characteristics, and applications of β-glucan phosphorylases. • β-Glucan phosphorylases in the production of functional carbohydrates.

UDP-Glycosyltransferases from the UGT344 Family Are Involved in Sulfoxaflor Resistance in Aphis gossypii Glover

UDP-glycosyltransferases (UGTs) are major phase II detoxification enzymes that catalyze the transfer of glycosyl residues from activated nucleotide sugars to acceptor hydrophobic molecules and play very important roles in the biotransformation of various endogenous and exogenous compounds. Our previous studies demonstrated that UGTs participated in the detoxification of insecticides in Aphis gossypii. However, the potential roles of UGTs in A. gossypii resistance to sulfoxaflor are still unclear. In this study, two inhibitors of UGT enzymes, sulfinpyrazone and 5-nitrouracil, significantly increased the toxicity of sulfoxaflor to a resistant strain of A. gossypii, whereas there were no synergistic effects in the susceptible strain. Based on the transcriptome sequencing results, the expression levels of 15 UGTs were analyzed by quantitative real-time PCR, and we found that seven UGT genes were highly over-expressed in a sulfoxaflor-resistant strain compared to the susceptible strain, including UGT344B4, UGT344C5, UGT344A11, UGT344A14, and UGT344L2. Further suppressing the expression of UGT344B4, UGT344C5, and UGT344A11 by RNA interference significantly increased the sensitivity of resistant aphids to sulfoxaflor, indicating that the overexpression of UGT genes is potentially associated with sulfoxaflor resistance. These results could provide valuable information for further understanding the mechanisms of insecticide resistance.

Nucleotide sugar biosynthesis occurs in the glycosomes of procyclic and bloodstream form Trypanosoma brucei

In Trypanosoma brucei, there are fourteen enzymatic biotransformations that collectively convert glucose into five essential nucleotide sugars: UDP-Glc, UDP-Gal, UDP-GlcNAc, GDP-Man and GDP-Fuc. These biotransformations are catalyzed by thirteen discrete enzymes, five of which possess putative peroxisome targeting sequences. Published experimental analyses using immunofluorescence microscopy and/or digitonin latency and/or subcellular fractionation and/or organelle proteomics have localized eight and six of these enzymes to the glycosomes of bloodstream form and procyclic form T. brucei, respectively. Here we increase these glycosome localizations to eleven in both lifecycle stages while noting that one, phospho-N-acetylglucosamine mutase, also localizes to the cytoplasm. In the course of these studies, the heterogeneity of glycosome contents was also noted. These data suggest that, unlike other eukaryotes, all of nucleotide sugar biosynthesis in T. brucei is compartmentalized to the glycosomes in both lifecycle stages. The implications are discussed.