Nucleotide Diphosphate Sugars Research Articles

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.

Development and Validation of a Universal High-Throughput UDP-Glycosyltransferase Assay with a Time-Resolved FRET Signal

Glycosyltransferase enzymes play diverse metabolic and regulatory roles by catalyzing the transfer of sugar molecules to protein, lipid, and carbohydrate acceptors, and they are increasingly of interest as therapeutic targets in a number of diseases, including metabolic disorders, cancer, and infectious diseases. The glycosyltransferases are a challenging target class from an assay development perspective because of the diversity of both donor and acceptor substrates and the lack of suitable glycan detection methods. However, many glycosyltransferases use uridine 5'-diphosphate (UDP) sugars as donor substrates, and detection of the free UDP reaction product provides a generic approach for measuring the activity of those enzymes. To exploit this approach for a broadly applicable high-throughput screening (HTS) assay for discovery of glycosyltransferase inhibitors, we developed a Transcreener(®) assay for immunodetection of UDP with a time-resolved Förster resonance energy transfer (TR-FRET) signal. We optimized the assay for detection of glycosyltransferase activity with nucleotide diphosphate (NDP) sugars at concentrations from 10 μM to 1 mM, achieving Z' values of 0.6 or higher. The assay was validated by orthogonal pooled screening with 8,000 compounds using polypeptide N-acetylgalactosaminyltransferase T3 as the target, and the hits were confirmed using an orthogonal readout. The reagents and signal were both stable for more than 8 h at room temperature, insuring robust performance in automated HTS environments. The TR-FRET-based UDP detection assay provides a broadly applicable approach for screening glycosyltransferases that use a UDP-sugar donor.

Analysis of nucleotide diphosphate sugar dehydrogenases reveals family and group-specific relationships.

UDP‐glucose dehydrogenase (UDPGDH), UDP‐N‐acetyl‐mannosamine dehydrogenase (UDPNAMDH) and GDP‐mannose dehydrogenase (GDPMDH) belong to a family of NAD +‐linked 4‐electron‐transfering oxidoreductases called nucleotide diphosphate sugar dehydrogenases (NDP‐SDHs). UDPGDH is an enzyme responsible for converting UDP‐d‐glucose to UDP‐d‐glucuronic acid, a product that has different roles depending on the organism in which it is found. UDPNAMDH and GDPMDH convert UDP‐N‐acetyl‐mannosamine to UDP‐N‐acetyl‐mannosaminuronic acid and GDP‐mannose to GDP‐mannuronic acid, respectively, by a similar mechanism to UDPGDH. Their products are used as essential building blocks for the exopolysaccharides found in organisms like Pseudomonas aeruginosa and Staphylococcus aureus. Few studies have investigated the relationships between these enzymes. This study reveals the relationships between the three enzymes by analysing 229 amino acid sequences. Eighteen invariant and several other highly conserved residues were identified, each serving critical roles in maintaining enzyme structure, coenzyme binding or catalytic function. Also, 10 conserved motifs that included most of the conserved residues were identified and their roles proposed. A phylogenetic tree demonstrated relationships between each group and verified group assignment. Finally, group entropy analysis identified novel conservations unique to each NDP‐SDH group, including residue positions critical to NDP‐sugar substrate interaction, enzyme structure and intersubunit contact. These positions may serve as targets for future research.Enzymes UDP‐glucose dehydrogenase (UDPGDH, EC 1.1.1.22).

Expanded acceptor substrates flexibility study of flavonol 7-O-rhamnosyltransferase, AtUGT89C1 from Arabidopsis thaliana

Acceptor substrates flexibility of previously characterized flavonol 7-O-rhamnosyltransferase (AtUGT89C1) from Arabidopsis thaliana was explored with an endogenous nucleotide diphosphate sugar and five different classes of flavonoids (flavonols, flavones, flavanones, chalcone and stilbenes) through a biotransformation approach. In contrast to the previous reports, this study highlights the expanded acceptor substrate promiscuity of AtUGT89C1 for the regiospecific glycosylation of diverse class of flavonoids at 7-hydroxyl position using microbial thymidine diphosphate (TDP)-l-rhamnose as sugar donor instead of uridine diphosphate-l-rhamnose. We examine the biocatalytic potential of AtUGT89C1 using endogenous sugar (TDP-l-rhamnose) from E. coli to generate a library of flavonoid 7-O-rhamnosides.

Design of Glycosyltransferase Inhibitors: Serine Analogues as Pyrophosphate Surrogates?

Mimicking the diphosphate moiety of nucleotide diphosphate sugars with serine analogues provided modest glycosyltransferase inhibitors. The synthetic strategy employed a combination of glycosylation, amide bond formation and azide-alkyne "click" chemistry. Inhibition constants (Ki ) in the high micromolar range were obtained with a selection of five galactosyltransferases. Cocrystals of three inhibitors bound at the active site of a blood group A/B synthesizing glycosyltransferase were analysed. The structures and inhibitory patterns of the analogues demonstrate the flexibility of the enzymes which complicates the rational design of glycosyltransferase inhibitors.

Enzymatic Biosynthesis of Novel Resveratrol Glucoside and Glycoside Derivatives.

A UDP glucosyltransferase from Bacillus licheniformis was overexpressed, purified, and incubated with nucleotide diphosphate (NDP) d- and l-sugars to produce glucose, galactose, 2-deoxyglucose, viosamine, rhamnose, and fucose sugar-conjugated resveratrol glycosides. Significantly higher (90%) bioconversion of resveratrol was achieved with α-d-glucose as the sugar donor to produce four different glucosides of resveratrol: resveratrol 3-O-β-d-glucoside, resveratrol 4'-O-β-d-glucoside, resveratrol 3,5-O-β-d-diglucoside, and resveratrol 3,5,4'-O-β-d-triglucoside. The conversion rates and numbers of products formed were found to vary with the other NDP sugar donors. Resveratrol 3-O-β-d-2-deoxyglucoside and resveratrol 3,5-O-β-d-di-2-deoxyglucoside were found to be produced using TDP-2-deoxyglucose as a donor; however, the monoglycosides resveratrol 4'-O-β-d-galactoside, resveratrol 4'-O-β-d-viosaminoside, resveratrol 3-O-β-l-rhamnoside, and resveratrol 3-O-β-l-fucoside were produced from the respective sugar donors. Altogether, 10 diverse glycoside derivatives of the medically important resveratrol were generated, demonstrating the capacity of YjiC to produce structurally diverse resveratrol glycosides.

Isolation of Sporothrix schenckii GDA1 and functional characterization of the encoded guanosine diphosphatase activity

Sporothrix schenckii is a fungal pathogen of humans and the etiological agent of sporotrichosis. In fungi, proper protein glycosylation is usually required for normal composition of cell wall and virulence. Upon addition of precursor oligosaccharides to nascent proteins in the endoplasmic reticulum, glycans are further modified by Golgi-glycosyl transferases. In order to add sugar residues to precursor glycans, nucleotide diphosphate sugars are imported from the cytosol to the Golgi lumen, the sugar is transferred to glycans, and the resulting nucleoside diphosphate is dephosphorylated by the nucleoside diphosphatase Gda1 before returning to cytosol. Here, we isolated the open reading frame SsGDA1 from a S. schenckii genomic DNA library. In order to confirm the function of SsGda1, we performed complementation assays in a Saccharomyces cerevisiae gda1∆ null mutant. Our results indicated that SsGDA1 restored the nucleotide diphosphatase activity to wild-type levels and therefore is a functional ortholog of S. cerevisiae GDA1.

Selective derivatization of nucleotide diphosphate (NDP)-4-keto sugars for electrospray ionization-mass spectrometry (ESI-MS)

Nucleotide diphosphate (NDP) sugars are widely present in antibiotics and glycoconjugates, such as protein- and lipid-linked oligosaccharides, where they act as substrates for glycosyltransferase in eukaryotes and prokaryotes. Among NDP sugars, NDP-4-keto sugars are key intermediates in the synthesis of structurally diverse NDP sugars with different functional groups. However, the structural identification of the NDP-4-keto sugars via mass spectrometry (electrospray ionization-mass spectrometry (ESI-MS)) continues to be a challenge because of the carbonyl group in these sugars interferes with ionization process. In this study, we evaluated various hydroxylamine compounds for the derivatization of NDP-4-keto sugars, so that the detection of the sugars by ESI-MS is more efficient. As a result, O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine was found to be the most effective tagging molecule for the detection of NDP-4-keto sugars without being interfered by original MS. This method can be used for identifying NDP-4-keto sugars such as thymidine diphosphate (TDP)-, adenosine diphosphate (ADP)-, uridine diphosphate (UDP)-, and cytosine diphosphate (CDP)-4-keto sugars as well as new NDP-4-keto-dehydratases.

Role of the Carbohydrate Binding Site of the Streptococcus pneumoniae Capsular Polysaccharide Type 3 Synthase in the Transition from Oligosaccharide to Polysaccharide Synthesis

The type 3 synthase catalyzes the formation of the Streptococcus pneumoniae type 3 capsular polysaccharide [-3)-beta-D-GlcUA-(1, 4)-beta-D-Glc-(1-]n. Synthesis is comprised of two distinct catalytic phases separated by a transition step whereby an oligosaccharylphosphatidylglycerol primer becomes tightly bound to the carbohydrate acceptor recognition site of the synthase. Using the recombinant synthase in Escherichia coli membranes, we determined that a critical oligosaccharide length of approximately 8 monosaccharides was required for recognition of the growing chain by the synthase. Upon binding of the oligosaccharide-lipid to the carbohydrate recognition site, the polymerization reaction entered a highly processive phase to produce polymer of high molecular weight. The initial oligosaccharide-synthetic phase also appeared to be processive, the duration of which was enhanced by the concentration of UDP-GlcUA and diminished by an increase in temperature. The overall reaction approached a steady state equilibrium between the polymer- and oligosaccharide-forming phases that was shifted toward the former by higher UDP-GlcUA levels or lower temperatures and toward the latter by lower concentrations of UDP-GlcUA or higher temperatures. The transition step between the two enzymatic phases demonstrated cooperative kinetics, which is predicted to reflect a possible reorientation of the oligosaccharide-lipid in conjunction with the formation of a tight binding complex.

N-Glycosylation of proteins by homogenate preparations of developing chick embryos.

Homogenate preparations of chick embryos contain the enzymes and endogenous precursors required for protein N-glycosylation mediated by dolichyl phosphate. The rate and extent of protein N-glycosylation was relatively high. Sensitivity of the first step in the dolichyl phosphate cycle to tunicamycin was confirmed. That tunicamycin had no indirect effect on the incorporation of glucose into lipid-linked intermediates or into protein but did reduce mannose incorporation was interpreted as supporting the view that the final glucosylation of the oligosaccharide is rate-limiting. Endogenous nucleotide diphosphate sugars and sugar acceptors, both lipid and protein linked, were present in the membrane preparation. Studies using dodecylsulphate-polyacrylamide gel electrophoresis on the radioactive glycoproteins formed during the incubations with nucleoside diphosphate [14C]sugars indicated that approximately one third of the 43 proteins detected carried radioactivity. Two thirds of the glycoproteins formed carried radioactive oligosaccharide chains that were either fully glucosylated or had been subsequently trimmed and received peripheral residues of N-acetylglucosamine. Most of the glycoproteins also carried radioactive oligosaccharide chains at varying stages of glycosylation preceding glucosylation and some carried only these ‘immature7rsquo; oligosaccharides. The transfer of mannose to protein and lipids was most active at 7 or 9 days of development being less active in both younger and older embryos whereas the pattern of change of activities of transfer of glucose and N-acetylglucosamine was more complicated. Most of the radioactive glycoproteins prominent at day 9 of development were also prominent in younger or older embryos. However, the incorporation of radioactive sugars into some glycoproteins, relative to that incorporated generally was very dependent upon the age of the embryos used.

Efficient chemical synthesis of both anomers of ADP l- glycero- and d- glycero-d- manno-heptopyranose

A series of anomeric phosphates and ADP-activated l- glycero- and d- glycero- d- manno-heptopyranoses has been prepared in high overall yields, which provided model compounds and substrates in the elucidation of biosynthetic pathways and glycosyl transfer reactions of nucleotide-activated bacterial heptoses. The α-anomers of the heptosyl phosphates were obtained in high yield and selectivity using the phosphoramidite procedure, whereas the β-phosphates were formed preferentially employing acylation of reducing heptoses with diphenyl phosphorochloridate. An efficient route to the formation of the nucleotide diphosphate sugars was elaborated by coupling of the O-acetylated phosphates with AMP-morpholidate followed by alkaline deprotection to furnish ADP- l- and d- glycero-α- d- manno-heptose in 84 and 89% yield, respectively. Deacetylation of the O-acetylated β-configured ADP heptoses was conducted at strictly controlled conditions (−28 °C at pH 10.5) to suppress formation of cyclic heptose-1,2-phosphodiesters with concomitant release of AMP. Isolation of the unstable β-configured ADP-heptoses by anion-exchange chromatography and gel-filtration afforded ADP l- and d- glycero-β- d- manno-heptose in high yields.

NDP‐sugars, floridoside and floridean starch levels in relation to activities of UDP‐glucose pyrophosphorylase and UDP‐glucose‐4‐epimerase in Solieria chorda l is (Rhodophyceae) under experimental conditions

SUMMARYUnder limited nutrient availability (i.e. unenriched sea‐water) and under 75 mol photons m–2 s–1 irradiance 12:12 LD, thalli of Solieria chordalis J. Agardh accumulated floridean starch and floridoside. When they were transferred into nutrient‐enriched seawater (150 umol L−1 NO31‐ and 7 umol L−1 P043i at 35 umol photons m−2 s−1 in irradiance 12:12 LD, starch and floridoside levels decreased. The main nucleotide diphosphate (NDP) sugars (i.e. UDP‐glucose, UDP‐galactose and ADP‐glucose) and the activities of UDP‐glucose pyrophosphorylase [Enzyme Code (EC) 2.7.7.9] and UDP‐glucose‐4‐epimerase (EC 5.1.3.2) were measured under these controlled culture conditions. Both UDP‐glucose and UDP‐galactose in the thal l i increased under conditions known to favor the accumulation of floridean starch and floridoside, whereas they decreased under conditions leading to floridean starch and floridoside breakdown. On the other hand, ADP‐glucose level only varied slightly. Although UDP‐glucose pyrophosphorylase activity rose under conditions of floridean starch synthesis, little variation was observed in UDP‐glucose‐4‐epimerase activity. These results suggest a possible enzymatic regulation of the NDP‐sugar and carbohydrate pool in which UDP‐glucose pyrophosphorylase would play a major role.

Glycosyl Fluorides Can Function as Substrates for Nucleotide Phosphosugar-dependent Glycosyltransferases

alpha-Galactosyl fluoride is shown to function as a substrate, in place of uridine-5'-diphosphogalactose, for the alpha-galactosyltransferase from Neisseria meningitidis. The reaction only occurs in the presence of catalytic quantities of uridine 5'-diphosphate. In the presence of galactosyl acceptors, the expected oligosaccharide product is formed in essentially quantitative yields, reaction having been performed on multi-milligram scales. In the absence of a suitable acceptor, the enzyme synthesizes uridine-5'-diphosphogalactose, as demonstrated through a coupled assay in which uridine-5'-diphosphogalactose is converted to uridine-5'-diphosphoglucuronic acid with conversion of NAD to NADH. These glycosyl fluoride substrates therefore offer the potential of an inexpensive alternative donor substrate in the synthesis of oligosaccharides as well a means of generating steady state concentrations of nucleotide diphosphate sugars for in situ use by other enzymes. Further, they should prove valuable as mechanistic probes.

Purification of 1,3-β-Glucan Synthase from Neurospora crassa by Product Entrapment

Awald, P., Zugel, M., Monks, C., Frost, D., and Selitrennikoff, C. P. 1993. Purification of 1,3-β-glucan synthase from Neurospora crassa by product entrapment. Experimental Mycology, 17, 130-141. 1,3-β-Glucan synthase activity of the ascomycete Neurospora crassa was purified ∼700-fold from hyphae. Hyphae were disrupted by bead-beating, and membrane-enriched fractions were obtained by high-speed centrifugation. Membranes were treated with (3-[(3-cholamidopropyl)dimethyl-ammoniol]I-propanesulfonate) and octyl-β-D-glucoside to solubilize enzyme activity. Soluble glucan synthase activity was incubated with substrate (UDP-glucose) and purified by centrifugation of enzyme associated with glucan (product entrapment). Purification was specific for UDP-glucose, the optimal concentration being 0.25 mM; no other nucleotide diphosphate sugar was able to significantly product-entrap enzyme activity. Partially purified enzyme activity formed β(1,3)-linked glucan, had a mean specific activity of 1900 nmol glucose incorporated/min/mg protein, a Km,app of 0.7 mM, and a Vmax of 0.5 nmol glucose incorporated/min. Separation of partially purified enzyme activity by SDS-PAGE showed a number of proteins copurifying with enzyme activity; computer analysis of digitized gel images revealed that proteins of 21, 25, 28, 45, 53, and 78 kDa were enriched. These results reinforce the view that 1,3-β-glucan synthase activity of fungi is a multimeric enzyme.

Synthesis and properties of 5-azido-UDP-glucose: Development of photoaffinity probes for nucleotide diphosphate sugar binding sites

Abstract A new active site directed photoaffinity probe, which is a model compound for studying nucleotide diphosphate sugar binding proteins, has been synthesized by coupling 5-azido-UTP and [32P]Glc-1-P using yeast UDP-glucose pyrophosphorylase to produce [beta-32P]5-azidouridine 5'-diphosphoglucose (5N3UDP-Glc). This probe has photochemical properties similar to that of 5-azidoUTP (Evans, R. K., and Haley, B. E. (1987) Biochemistry 26, 269-276). The efficacy of 5N3UDP-Glc as an active site directed probe was demonstrated using yeast UDP-Glc pyrophosphorylase. Saturation effects of photoinsertion were observed with an apparent Kd of 51 microM and the natural substrate, UDP-Glc, prevented photoinsertion of [beta-32P]5N3UDP-Glc with an apparent Kd of 87 microM. Prevention of photoinsertion was also seen with UTP and pyrophosphate with apparent Kd values less than 200 microM. UMP, UDP, ATP, and GTP were much less effective competitors. Selective photoinsertion was observed with several partially purified enzymes including UDP-Glc dehydrogenase, UDP-Gal-4-epimerase, Gal-1-P uridyltransferase, and phosphorylase a. The absence of nonselective photoinsertion into bulk proteins was demonstrated with crude homogenates of rabbit liver as well as with several UDP-Glc binding proteins. Of the six purified enzymes tested, only phosphoglucomutase has been shown to incorporate radiolabel from the photoprobe in the absence of UV irradiation. These results and a discussion of the utility of 5N3UDP-Glc for detecting UDP-Glc binding proteins and isolating active site peptides are presented.

Behavior of sugar derivatives in procedures for ganglioside isolation.

A common method of studying ganglioside metabolism is to measure the amounts of radioactivity incorporated into ganglioside from a radiolabeled precursor. This requires that radioactive nonganglioside material be completely removed from the ganglioside fraction. Nucleotide sugars and aminosugars comprise an important source of such contaminants. Therefore, we have studied their behaviors in several procedures currently employed to isolate gangliosides. Over 50% of the radioactivity associated with several nucleotide sugars added to a brain homogenate is extracted with chloroform/methanol (2:1, v/v), and most of this is recovered in the upper phase of a Folch partition. Dialysis against water removes almost all of the free aminosugar but only 70% of nucleotide sugar. Treatment with alkaline phosphatase, phosphodiesterase and alkaline methanol followed by dialysis removes almost all of the nucleotide diphosphate sugars but only 88% of cytidine 5'-monophosphate sialic acid (CMP-NeuAc). Nucleotide sugars cannot be separated from gangliosides by Unisil or Iatrobead chromatography, but nucleotide diphosphate sugars and gangliosides are resolved with Sephadex LH-20 chromatography following treatment with phosphodiesterase and alkaline phosphatase. CMP-NeuAc was not satisfactorily separated from gangliosides using any of the procedures.

On the stability of nucleotide and nucleoside diphosphate sugar concentrations in isolated hepatocytes [proceedings

On the stability of nucleotide and nucleoside diphosphate sugar concentrations in isolated hepatocytes [proceedings

The metal ion catalyzed decomposition of nucleoside diphosphate sugars.

The metal ion catalysed decomposition of the nucleotide diphosphate sugars, uridine diphosphate glucose, uriding diphosphate galactose, uridine diphosphate N-acetylglucosamine, guanosine diphosphate mannose, and guanosine diphosphate fucose (UDPGlc, UDPGal, UDPGlc-NAc, GDPMan, and GDPFuc, respectively), has been studies as a function of pH. UDPDlc and UDPGal decompose readily to the a,2-cycle phosphate derivative of the sugar and uridine 5'-phosphoric acid (UMP) in the presence of Mn2+. Under all conditions tested, UDPGal decomposes two to three times more rapidly than does UDPGlc. GDPFuc is slowly degraded to free fucose under similar conditions; the other nucleotide diphosphate sugars are stable. The rate of reaction increases with increasing hydroxide ion concentration from pH 6.5 to 7.9 and with metal ion concentration from 10 to 200 mm. Several metal ions are effective catalysts; at pH 7.5 WITH 20 mM UDPGal and 20 mM metal ion, the following apparent first-order rate constants (min-1 x 10(4)) were obtained: Eu3+ 700; Mn2+, 70; Co2+ 27; Zn2+, 22; Ca2+, 3.0; Cu2+, 2.4; and Mg2+, 0. It appears that Mn2+ concentrations that have been used in studies with nucleotide diphosphate sugars at neutral pH can catalyze significant decomposition leading to erroneous interpretation of kinetic and incorporation experiments.

Glycoprotein biosynthesis: purification and properties of glycoprotein. N-acetylglucosaminyl transferases from guinea pig liver utilizing endogenous and exogenous acceptors.

A glycoprotein :N‐acetylglucosamine transferase utilizing endogenous acceptors and a fetuin: N‐acetylglucosamine transferase utilizing a defined exogenous fetuin acceptor from which sialic acid, galactose and N‐acetylglucosamine had been removed were characterized from guinea pig liver. Both glycoprotein :N‐acetylglucosamine systems used UDP‐N‐acetylglucosamine as substrate, required Mn++ and had optimum pH of 5.4. Both enzyme systems were solubilized by extraction of the liver with 0.1% Triton X‐100. The fetuin :N‐acetylglucosamine transferase was separated from endogenous acceptors and purified more than 24‐fold on Sephadex G‐200. The endogenous system had a Km of 0.6 μM and the exogenous fetuin :N‐acetylglucosamine transferase had a Km of 3.8 μM with respect to N‐[14C]acetylglucosamine. The fetuin :N‐acetylglucosamine transferase was highly specific; 11 glycoproteins and 20 mono‐ and disaccharides and amino acids were negative when tested for acceptor activity. The fetuin :N‐acetylglucosamine transferase did not transfer any other nucleotide diphosphate sugar onto the acceptor; even UDP‐N‐acetylgalactosamine was not transferred. It appeared that the exogenous and endogenous systems may well be identical except that the endogenous acceptor was removed during purification of the enzyme.

Nucleotide diphosphate sugar pyrophosphatase activity in the skin of alloxan-diabetic rats

Nucleotide diphosphate sugar pyrophosphatase activity in the skin of alloxan-diabetic rats