Diphosphate Sugars Research Articles

Proteome-wide bioinformatic annotation and functional validation of the monotopic phosphoglycosyl transferase superfamily

Phosphoglycosyl transferases (PGTs) are membrane proteins that initiate glycoconjugate biosynthesis by transferring a phospho-sugar moiety from a soluble nucleoside diphosphate sugar to a membrane-embedded polyprenol phosphate acceptor. The centrality of PGTs in complex glycan assembly and the current lack of functional information make these enzymes high-value targets for biochemical investigation. In particular, the small monotopic PGT family is exclusively bacterial and represents the minimal functional unit of the monotopic PGT superfamily. Here, we combine a sequence similarity network analysis with a generalizable, luminescence-based activity assay to probe the substrate specificity of this family of monoPGTs in the bacterial cell-membrane fraction. This strategy allows us to identify specificity on a far more significant scale than previously achievable and correlate preferred substrate specificities with predicted structural differences within the conserved monoPGT fold. Finally, we present the proof-of-concept for a small-scale inhibitor screen (eight nucleoside analogs) with four monoPGTs of diverse substrate specificity, thus building a foundation for future inhibitor discovery initiatives.

Hypothetical protein CuvA (Rv1422) from Mycobacterium tuberculosis H37Rv interacts with uridine diphosphate N-acetylglucosamine as a key precursor of cell wall

Mycobacterium tuberculosis CuvA (Rv1422, MtCuvA) has previously been suggested that it may play a critical role in nutrient utilization and cell wall synthesis required for physiological adaptation in a host cell, but its biochemical details remain unclear. Our previous studies showed that MtCuvA can bind to uridine diphosphate (UDP) sugars as a cell wall precursor component. To verify its functional roles, we report here the biochemical properties of MtCuvA for the binding of UDP-N-acetylglucosamine (GlcNAc) using site-directed mutagenesis and docking simulation. The KD values for UDP-sugars indicate that MtCuvA prefers to bind UDP-GlcNAc as a physiological ligand compared to UDP-glucose. Mutational studies of MtCuvA showed that H12A, T33A, D36A, Q154A, S196, T199A, N226A, and H298A mutants significantly affected the binding to UDP-GlcNAc. We also observed that UDP, but not GlcNAc, could bind to MtCuvA. These results imply that the presence of UDP moiety in the ligand is necessary for interaction with MtCuvA. Moreover, mutational studies of MtCuvA with UDP showed that residues H12, S196, T199, N226, and H298 may be involved in its binding to the UDP moiety, almost consistent with the docking simulation results. Our results provide an insight into the interaction of MtCuvA with UDP-GlcNAc as a key precursor of peptidoglycan.

Glycosylated queuosines in tRNAs optimize translational rate and post-embryonic growth

Transfer RNA (tRNA) modifications are critical for protein synthesis. Queuosine (Q), a 7-deaza-guanosine derivative, is present in tRNA anticodons. In vertebrate tRNAs for Tyr and Asp, Q is further glycosylated with galactose and mannose to generate galQ and manQ, respectively. However, biogenesis and physiological relevance of Q-glycosylation remain poorly understood. Here, we biochemically identified two RNA glycosylases, QTGAL and QTMAN, and successfully reconstituted Q-glycosylation of tRNAs using nucleotide diphosphate sugars. Ribosome profiling of knockout cells revealed that Q-glycosylation slowed down elongation at cognate codons, UAC and GAC (GAU), respectively. We also found that galactosylation of Q suppresses stop codon readthrough. Moreover, protein aggregates increased in cells lacking Q-glycosylation, indicating that Q-glycosylation contributes to proteostasis. Cryo-EM of human ribosome-tRNA complex revealed the molecular basis of codon recognition regulated by Q-glycosylations. Furthermore, zebrafish qtgal and qtman knockout lines displayed shortened body length, implying that Q-glycosylation is required for post-embryonic growth in vertebrates.

Structural Characterization and Ligand-Induced Conformational Changes of SenB, a Se-Glycosyltransferase Involved in Selenoneine Biosynthesis.

Selenium (Se) is an essential micronutrient that is found naturally in proteins, nucleic acids, and natural products. Unlike selenoproteins and selenonucleic acids, little is known about the structures of biosynthetic enzymes that incorporate Se into small molecules. Here, we report the X-ray crystal structure of SenB, the first known Se-glycosyltransferase that was recently found to be involved in the biosynthesis of the Se-containing metabolite selenoneine. SenB catalyzes C-Se bond formation using selenophosphate and an activated uridine diphosphate sugar as a Se and glycosyl donor, respectively, making it the first known selenosugar synthase and one of only four bona fide C-Se bond-forming enzymes discovered to date. Our crystal structure, determined to 2.25 Å resolution, reveals that SenB is a type B glycosyltransferase, displaying the prototypical fold with two globular Rossmann-like domains and a catalytic interdomain cleft. By employing complementary structural biology techniques, we find that SenB undergoes both local and global substrate-induced conformational changes, demonstrating a significant increase in α-helicity and a transition to a more compact conformation. Our results provide the first structure of SenB and set the stage for further biochemical characterization in the future.

Anaerobic dissimilatory phosphite oxidation, an extremely efficient concept of microbial electron economy.

Phosphite is a stable phosphorus compound that, together with phosphate, made up a substantial part of the total phosphorus content of the prebiotic Earth's crust. Oxidation of phosphite to phosphate releases electrons at an unusually low redox potential (-690 mV at pH 7.0). Numerous aerobic and anaerobic bacteria use phosphite as a phosphorus source and oxidise it to phosphate for synthesis of nucleotides and other phosphorus-containing cell constituents. Only two pure cultures of strictly anaerobic bacteria have been isolated so far that use phosphite as an electron donor in their energy metabolism, the Gram-positive Phosphitispora fastidiosa and the Gram-negative Desulfotignum phosphitoxidans. The key enzyme of this metabolism is an NAD+ -dependent phosphite dehydrogenase enzyme that phosphorylates AMP to ADP. These phosphorylating phosphite dehydrogenases were found to be related to nucleoside diphosphate sugar epimerases. The produced NADH is channelled into autotrophic CO2 fixation via the Wood-Ljungdahl (CO-DH) pathway, thus allowing for nearly complete assimilation of the substrate electrons into bacterial biomass. This extremely efficient type of electron flow connects energy and carbon metabolism directly through NADH and might have been important in the early evolution of life when phosphite was easily available on Earth.

Feedback Inhibition of Bacterial Nucleotidyltransferases by Rare Nucleotide l-Sugars Restricts Substrate Promiscuity.

Bacterial glycomes are rich in prokaryote-specific or "rare" sugars that are absent in mammals. Like common sugars found across organisms, rare sugars are typically activated as nucleoside diphosphate sugars (NDP-sugars) by nucleotidyltransferases. In bacteria, the nucleotidyltransferase RmlA initiates the production of several rare NDP-sugars, which in turn regulate downstream glycan assembly through feedback inhibition of RmlA via binding to an allosteric site. In vitro, RmlA activates a range of common sugar-1-phosphates to produce NDP-sugars for biochemical and synthetic applications. However, our ability to probe bacterial glycan biosynthesis is hindered by limited chemoenzymatic access to rare NDP-sugars. We postulate that natural feedback mechanisms impact nucleotidyltransferase utility. Here, we use synthetic rare NDP-sugars to identify structural features required for regulation of RmlA from diverse bacterial species. We find that mutation of RmlA to eliminate allosteric binding of an abundant rare NDP-sugar facilitates the activation of noncanonical rare sugar-1-phosphate substrates, as products no longer affect turnover. In addition to promoting an understanding of nucleotidyltransferase regulation by metabolites, this work provides new routes to access rare sugar substrates for the study of important bacteria-specific glycan pathways.

Expanding the Substrate Scope of a Bacterial Nucleotidyltransferase via Allosteric Mutations.

Bacterial glycoconjugates, such as cell surface polysaccharides and glycoproteins, play important roles in cellular interactions and survival. Enzymes called nucleotidyltransferases use sugar-1-phosphates and nucleoside triphosphates (NTPs) to produce nucleoside diphosphate sugars (NDP-sugars), which serve as building blocks for most glycoconjugates. Research spanning several decades has shown that some bacterial nucleotidyltransferases have broad substrate tolerance and can be exploited to produce a variety of NDP-sugars in vitro. While these enzymes are known to be allosterically regulated by NDP-sugars and their fragments, much work has focused on the effect of active site mutations alone. Here, we show that rational mutations in the allosteric site of the nucleotidyltransferase RmlA lead to expanded substrate tolerance and improvements in catalytic activity that can be explained by subtle changes in quaternary structure and interactions with ligands. These observations will help inform future studies on the directed biosynthesis of diverse bacterial NDP-sugars and downstream glycoconjugates.

Backbone-Anchoring, Solid-Phase Synthesis Strategy To Access a Library of Peptidouridine-Containing Small Molecules.

Nucleoside diphosphate sugar (NDP-sugar) substrates provide the inspiration for nucleoside analogue inhibitor scaffolds. By employing solid-phase synthesis, we provide a method to access a library of peptidouridine inhibitors with both minimal compound handling and purification steps. Specifically, this strategy is exemplified by generating uridine diphosphate sugar (UDP-sugar) mimics, which allow for compound elaboration by altering the dipeptide composition, the N-terminal linkage, and a pendant aryl group. To exemplify the versatility, 41 unique nucleoside analogues are presented.

Exploration of archaeal nucleotide sugar epimerases unveils a new and highly promiscuous GDP-Gal4E subgroup

• Promiscuity of phGal4E_1 on different NDP-sugars was explored. • First demonstration of C4 epimerization activity on GDP-L-galactose and GDP-L-fucose. • PhGal4E_1, afGal4E and pcGal4E could form a new subgroup within CEP1 C4-epimerases. • The heptagonal box model allows the identification of new specificities in CEP1 family enzymes. UDP-galactose 4-epimerase (Gal4E, EC 5.1.3.2) catalyses the interconversion of UDP-galactose and UDP-glucose by inverting the configuration of the 4’-hydroxyl group of the sugar moiety. This enzyme is one of the mechanistically best-characterized members of the Short-chain Dehydrogenase/Reductase (SDR) superfamily. Although Gal4E was widely studied throughout all domains of life, ranging from eukaryotes to archaea, its biochemical characterization was often limited to UDP-hexoses, neglecting the possibility that Gal4E might be promiscuous towards other NDP-sugars and derivatives thereof. In this study, we identified a novel Gal4E subgroup displaying an unprecedented specificity on guanosine diphosphate (GDP) sugars. As a proof of concept, a detailed biochemical investigation was performed on Gal4E from Pyrococcus horikoshii (phGal4E_1), which revealed that it has, in fact, a clear preference for GDP-sugars. In addition, we confirmed that it accepts a variety of other sugar moieties, including L-sugars like L-galactose and L-fucose.

Probing rapid carbon fixation in fast-growing seaweed Ulva meridionalis using stable isotope 13C-labelling

The high growth rate of Ulva seaweeds makes it a potential algal biomass resource. In particular, Ulva meridionalis grows up to fourfold a day. Here, we demonstrated strong carbon fixation by U. meridionalis using 13C stable isotope labelling and traced the 13C flux through sugar metabolites with isotope-ratio mass spectrometry (IR-MS), Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), 13C-nuclear magnetic resonance spectrometry (13C-NMR), and gas chromatography-mass spectrometry (GC–MS). U. meridionalis was first cultured in 13C-labelled enriched artificial seawater for 0–12 h, and the algae were collected every 4 h. U. meridionalis grew 1.8-fold (dry weight), and the 13C ratio reached 40% in 12 h, whereas 13C incorporation hardly occurred under darkness. At the beginning of the light period, 13C was incorporated into nucleic diphosphate (NDP) sugars in 4 h, and 13C labelled peaks were identified using FT-ICR-MS spectra. Using semiquantitative 13C-NMR measurements and GC–MS, 13C was detected in starch and matrix polysaccharides after the formation of NDP sugars. Moreover, the 14:10 light:dark regime resulted into 85% of 13C labelling was achieved after 72 h of cultivation. The rapid 13C uptake by U. meridionalis shows its strong carbon fixation capacity as a promising seaweed biomass feedstock.

Synthesis of Triazole-Containing Furanosyl Nucleoside Analogues and Their Phosphate, Phosphoramidate or Phoshonate Derivatives as Potential Sugar Diphosphate or Nucleotide Mimetics.

The synthesis of stable and potentially bioactive xylofuranosyl nucleoside analogues and potential sugar diphosphate or nucleotide mimetics comprising a 1,2,3-triazole moiety is reported. 3'-O-Methyl-branched N-benzyltriazole isonucleosides were accessed in 5-7 steps and 42-54 % overall yields using a Cu(I)-catalyzed cycloaddition of 3-O-propargyl-1,2-O-isopropylidene-α-D-xylofuranose with benzyl azide as key step. Related isonucleotides were obtained by 5-O-phosphorylation of acetonide-protected 3-O-propargyl xylofuranose and further "click" cycloaddition or by Staudinger-phosphite reaction of a 5-azido N-benzyltriazole isonucleoside. Hydroxy-, amino- or bromomethyl triazole 5'-isonucleosides were synthesized by thermal cycloaddition of 5-azido 3-O-benzyl/dodecyl xylofuranoses with propargyl alcohol, propargylamine or propargyl bromide. Better yields (82-85 %) were obtained when using propargyl alcohol and a high 1,4-regioselectivity was attained with propargyl bromide. Further O/N-phosphorylation or Arbuzov reaction led to (triazolyl)methyl phosphates, phosphoramidates or phosphonates. The latter were converted into uracil nucleoside 5'-(triazolyl)methyl phosphonates as prospective nucleoside diphosphate mimetics.

Utility of bacterial peptidoglycan recycling enzymes in the chemoenzymatic synthesis of valuable UDP sugar substrates.

Uridine diphosphate (UDP) sugars are essential precursors for glycosylation reactions in all forms of life. Reactions that transfer the carbohydrate from the UDP donor are catalyzed by glycosyltransferases (Gtfs). While the stereochemistry and negative physiological charge of UDP-sugars are essential for their biochemical function in the cell, these characteristics make them challenging molecules to synthesize and purify on scale in the laboratory. This chapter focuses on the utilization of a chemoenzymatic synthesis of muramyl UDP-sugars, key building blocks in the bacterial cell peptidoglycan. A scalable strategy to obtain UDP-N-acetyl muramic acid derivatives (UDP-NAM), the first committed intermediate used solely in peptidoglycan biosynthesis, is described herein. This methodology utilizes two enzymes involving the cell wall recycling enzymes MurNAc/GlcNAc anomeric kinase (AmgK) and NAM α-1-phosphate uridylyl transferase (MurU), respectively. The promiscuity of these enzymes allows for the unique chemical functionality to be embedded in bacterial peptidoglycan both in vitro and in whole bacterial cells for subsequent structural and functional studies of this important biopolymer.

A Review on Structure, Modifications and Structure-Activity Relation of Quercetin and Its Derivatives.

Quercetin and its derivatives are important metabolites that belong to the flavonol class of flavonoids. Quercetin and some of the conjugates have been approved by the FDA for human use. They are widely distributed among plants and have various biological activities, such as being anticancer, antiviral, and antioxidant. Hence, the biosynthesis of novel derivatives is an important field of research. Glycosylation and methylation are two important modification strategies that have long been used and have resulted in many novel metabolites that are not present in natural sources. A strategy for modifying quercetin in E. coli by means of glycosylation, for example, involves overexpressing respective glycosyltransferases (GTs) in the host and metabolic engineering for increasing nucleoside diphosphate sugar (NDP sugar). Still others have used microorganisms other than E. coli, such as Streptomyces sp., for the biotransformation process. The overall study of the structural activity relationship has revealed that modification of some residues in quercetin decreased one activity but increased others. This review summarizes all of the information mentioned above.

Solid-phase Synthesis of Phosphorus Derivatives

The solid-phase synthesis (SPS) of phosphorus-containing compounds is based mainly on the fact that the chemical process is conducted in a two-phase system. One of the components is connected via covalent bonds to a solid support, which is in general an insoluble polymer, representing the solid phase of the process. The other components involved into the process are solubilized in a solution. The method is suitable to be applied to almost any organic compounds. A common example of using solid-phase synthesis is for obtaining products nucleotide containing, similar to nucleic acids. During the whole process, the nucleotide is always on the solid phase, after the condensation reaction, except for the last step, when the synthesis is already finished. Then, the product is released and separated very easily by filtration. The obtained polymer-oligonucleotide product can participate further in condensation reactions as well. Other important biomolecules synthesized by solid-phase approach during the last decades are nucleoside di- and triphosphates, nucleoside diphosphate sugars and dinucleoside polyphosphates. Those products are precursors of deoxysugars, aminodeoxysugars, uronic acids or glycoconjugates, and are also necessary for DNA and RNA synthesis. The use of the solid-phase method in the context of immobilized oligomers is of great interest nowadays. The solid-phase synthesis offers many advantages in comparison with the conventional solution-phase method, because it takes much less time, it is highly stereoselective, the products are separated and purified usually by a simple filtration or decantation, solvents with high boiling points could be used, the whole process is based on solid polymer support and the obtained compounds should not be isolated.

Making Molecules with Clay: Layered Double Hydroxides, Pentopyranose Nucleic Acids and the Origin of Life.

A mixture of sugar diphosphates is produced in reactions between small aldehyde phosphates catalysed by layered double hydroxide (LDH) clays under plausibly prebiotic conditions. A subset of these, pentose diphosphates, constitute the backbone subunits of nucleic acids capable of base pairing, which is not the case for the other products of these LDH-catalysed reactions. Not only that, but to date no other polymer found capable of base pairing—and therefore information transfer—has a backbone for which its monomer subunits have a plausible prebiotic synthesis, including the ribose-5-phosphate backbone subunit of RNA. Pentose diphosphates comprise the backbone monomers of pentopyranose nucleic acids, some of the strongest base pairing systems so far discovered. We have previously proposed that the first base pairing interactions were between purine nucleobase precursors, and that these were weaker and less specific than standard purine-pyrimidine interactions. We now propose that the inherently stronger pairing of pentopyranose nucleic acids would have compensated for these weaker interactions, and produced an informational polymer capable of undergoing nonenzymatic replication. LDH clays might also have catalysed the synthesis of the purine nucleobase precursors, and the polymerization of pentopyranose nucleotide monomers into oligonucleotides, as well as the formation of the first lipid bilayers.

Synthesis of Phenoxyundecyl Diphosphate Disaccharides for Studies of the Biosynthesis of O Antigenic Polysaccharides in Enteric Bacteria.

The biosynthesis of O antigenic polysaccharides in enteric bacteria from nucleoside diphosphate sugars (donor substrates) is catalyzed by the corresponding glycosyltransferases and proceeds through the intermediate formation of undecaprenyl diphosphate sugars (acceptor substrates). To study this process, a chemical synthesis of the compounds having the natural structure or their modified analogs is necessary. The phosphoroimidazolidate method is a universal method for synthesis of lipid diphosphate disaccharides containing 2-acetamido-2-deoxyglycosyl residue at the reducing end of the disaccharide moiety and 11-phenoxyundecyl residue as lipid fragment of the molecule. We report here protocols to synthesize the disaccharides P1-(11-phenoxyundecyl)-P2-(2-acetamido-2-deoxy-3-O-α-D-rhamnopyranosyl-α-D-glucopyranosyl) diphosphate [D-Rha(α1-3)-D-GlcNAcα-PP-PhU, Compound 1] and P1-(11-phenoxyundecyl)-P2-(2-acetamido-2-deoxy-3-O-β-D-galactopyranosyl-α-D-galactopyranosyl) diphosphate [D-Gal(β1-3)-D-GalNAcα-PP-PhU, Compound 6]. We describe the procedures for identification and structure estimation of compounds by TLC, NMR, and MS. We also include the biochemical testing of Compound 6 with α2,3-sialyltransferase WbwA from Escherichia coli O104.

Synthesis of P1-(11-phenoxyundecyl)-P2-(2-acetamido-2-deoxy-3-O-α-D-rhamnopyranosyl-α-D-glucopyranosyl) diphosphate and P1-(11-phenoxyundecyl)-P2-(2-acetamido-2-deoxy-3-O-β-D-galactopyranosyl-α-D-galactopyranosyl) diphosphate for the investigation of biosynthesis of O-antigenic polysaccharides in Pseudomonas aeruginosa and Escherichia coli O104

Two new phenoxyundecyl diphosphate sugars were synthesized for the first time: P1-(11-phenoxyundecyl)-P2- (2-acetamido-2-deoxy-3-O-α-D-rhamnopyranosyl-α-D-glucopyranosyl) diphosphate and P1-(11-phenoxyundecyl)-P2-(2-acetamido-2-deoxy-3-O-β-D-galactopyranosyl-α-D-galactopyranosyl) diphosphate to study the third step of biosynthesis of the repeating units of O-antigenic polysaccharides in Pseudomonas aeruginosa and E.coli O104 respectively.

Bacterial phosphoglycosyl transferases: initiators of glycan biosynthesis at the membrane interface.

Phosphoglycosyl transferases (PGTs) initiate the biosynthesis of both essential and virulence-associated bacterial glycoconjugates including lipopolysaccharide, peptidoglycan and glycoproteins. PGTs catalyze the transfer of a phosphosugar moiety from a nucleoside diphosphate sugar to a polyprenol phosphate, to form a membrane-bound polyprenol diphosphosugar product. PGTs are integral membrane proteins, which include between 1 and 11 predicted transmembrane domains. Despite this variation, common motifs have been identified in PGT families through bioinformatics and mutagenesis studies. Bacterial PGTs represent important antibacterial and virulence targets due to their significant role in initiating the biosynthesis of key bacterial glycoconjugates. Considerable effort has gone into mechanistic and inhibition studies for this class of enzymes, both of which depend on reliable, high-throughput assays for easy quantification of activity. This review summarizes recent advances made in the characterization of this challenging but important class of enzymes.

On the stability of nucleoside diphosphate glucose metabolites: implications for studies of plant carbohydrate metabolism.

Nucleoside diphosphate sugars (NDP-sugars) are the substrates for biosynthesis of oligo- and polysaccharides, such as starch and cellulose, and are also required for biosynthesis of nucleotides, ascorbic acid, several cofactors, glycoproteins and many secondary metabolites. A controversial study that questions the generally accepted pathway of ADP-glucose and starch synthesis in plants is based, in part, on claims that NDP-sugars are unstable at alkaline pH in the presence of Mg2+ and that this instability can lead to unreliable results from in vitro assays of enzyme activities. If substantiated, this claim would have far-reaching implications for many published studies that report on the activities of NDP-sugar metabolizing enzymes. To resolve this controversy, we investigated the stability of UDP- and ADP-glucose using biophysical, namely nuclear magnetic resonance (NMR), and highly specific enzymatic methods. Results obtained with both techniques indicate that NDP-sugars are not as unstable as previously suggested. Moreover, their calculated in vitro half-lives are significantly higher than estimates of their in planta turnover times. This indicates that the physico-chemical stability of NDP-sugars has little impact on their concentrations in vivo and that NDP-sugar levels are determined primarily by the relative rates of enzymatic synthesis and consumption. Our results refute one of the main arguments for the controversial pathway of starch synthesis from imported ADP-glucose produced by sucrose synthase in the cytosol.

Application of synthetic lipid diphosphate sugars for studies of biosynthesis of O-antigenic polysaccharides of enteric bacteria

Application of synthetic lipid diphosphate sugars for studies of biosynthesis of O-antigenic polysaccharides of enteric bacteria