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Sugar Nucleotides (sugar + nucleotide)
Selected AbstractsFRET-Based Direct and Continuous Monitoring of Human Fucosyltransferases Activity: An Efficient synthesis of Versatile GDP- L -Fucose Derivatives from Abundant d- Galactose,CHEMISTRY - A EUROPEAN JOURNAL, Issue 2 2008Takahiro Maeda Abstract We have developed a facile and versatile protocol for the continuous monitoring of human fucosyltransferases activity by using fluorescence energy resonance transfer (FRET), and have explored the feasibility of its use in an inhibitor screening assay. A convenient sugar nucleotide with a fluorogenic probe, 6-deoxy-6- N -(2-naphalene-2-yl-acetamide)-,- L -galactopyranos-1-yl-guanosine 5,-diphosphate disodium salt (1), was efficiently synthesized from naturally abundant D -galactopyranose via a key intermediate, 6-azide-1,2,3,4-tetra- O -benzoyl-6-deoxy-,- L -galactopyranose (10). It was demonstrated that the combined use of the glycosyl donor 1 and a dansylated acceptor substrate, sialyl-,2,3-LacNAc derivative (2) allowed us to carry out highly sensitive, direct, and continuous in vitro monitoring of the generation of sialyl Lewis,X (SLex), which is catalyzed by human ,-1,3-fucosyltransferase,VI (FUT-VI). A kinetic analysis revealed that compound 1 was an excellent donor substrate (KM=0.94,,M and Vmax=0.14,,M,min,1) for detecting human FUT-VI activity. To the best of our knowledge, this synthetic fluorogenic probe is the most sensitive and selective donor substrate for FUT-VI among all of the known GDP-Fuc analogues, including the parent GDP-Fuc. When a dansylated asparagine-linked glycopeptide 20, which is derived from egg yolk was employed as an alternate acceptor substrate, a FRET-based assay with compound 1 could be used to directly monitor the ,1,6-fucosylation at the reducing terminal GlcNAc residue by human FUT-VIII (KM=175,,M and Vmax=0.06,,M/,min); this indicates that the present method might become a general protocol for the characterization of various mammalian fucosyltransferases in the presence of designated fluorogenic acceptor substrates. The present protocol revealed that compound 23, which was obtained by a 1,3-dipolar cycloaddition between the disodium salt 16 and 1-ethynyl-naphthalene exhibits highly potent inhibitory effects against the FUT-VI-mediated sialyl Lewis,X synthesis (IC50=5.4,,M). [source] Conserved cytoplasmic motifs that distinguish sub-groups of the polyprenol phosphate:N -acetylhexosamine-1-phosphate transferase familyFEMS MICROBIOLOGY LETTERS, Issue 2 2000Matt S. Anderson Abstract WecA, MraY and WbcO are conserved members of the polyprenol phosphate:N -acetylhexosamine-1-phosphate transferase family involved in the assembly of bacterial cell walls, and catalyze reactions involving a membrane-associated polyprenol phosphate acceptor substrate and a cytoplasmically located UDP- D -amino sugar donor. MraY, WbcO and WecA purportedly utilize different UDP-sugars, although the molecular basis of this specificity is largely unknown. However, domain variations involved in specificity are predicted to occur on the cytoplasmic side of the membrane, adjacent to conserved domains involved in the mechanistic activity, and with access to the cytoplasmically located sugar nucleotides. Conserved C-terminal domains have been identified that satisfy these criteria. Topological analyses indicate that they form the highly basic, fifth cytoplasmic loop between transmembrane regions IX and X. Four diverse loops are apparent, for MraY, WecA, WbcO and RgpG, that uniquely characterize these sub-groups of the transferase family, and a correlation is evident with the known or implied UDP-sugar specificity. [source] Antioxidative Enzymes and Sucrose Synthase Contribute to Cold Stress Tolerance in ChickpeaJOURNAL OF AGRONOMY AND CROP SCIENCE, Issue 5 2009S. Kaur Abstract Chickpea is sensitive to low temperature (<10°C) during its reproductive stage. Low temperature adversely affects the development of pods and seeds. This study was undertaken to investigate the role of sucrose metabolizing enzymes in seed development and potential of antioxidative enzymes in protecting seeds and podwalls from the deleterious effects of cold stress in advanced cold tolerant chickpea breeding lines. Healthy pod set was observed in these tolerant lines in the end of December where as low temperature susceptible PBG-1 did not flower. Two lines ICCV 96029 and ICCV 96030 showed susceptible characters such as reduced flowering, blackened and shrivelled seeds and yellowish pods in comparison to other cold stress tolerant lines due to sudden dip of temperature (<1 °C) during the first week of January. These two lines were, therefore, treated as susceptible checks in comparison to other tolerant lines. A significantly higher activity and specific activity of sucrose synthase was observed in seeds of most of the cold tolerant lines in comparison with ICCV 96029 and ICCV 96030, thereby providing sugars as well as sugar nucleotides for their growth and starch synthesis during unfavourable low temperature. The developing seeds and podwalls of tolerant genotypes had higher activities of antioxidant enzymes, i.e. catalase, ascorbate peroxidase and glutahione reductase in comparison with ICCV 96029 and ICCV 96030. It appears that the higher activities of antioxidant enzymes in podwall protect the developing seeds from cold stress. [source] Exopolysaccharide (EPS) biosynthesis by Lactobacillus sakei 0,1: production kinetics, enzyme activities and EPS yieldsJOURNAL OF APPLIED MICROBIOLOGY, Issue 3 2001B. Degeest Aims:,To determine optimal exopolysaccharide (EPS) production conditions of the mesophilic lactic acid bacterium strain Lactobacillus sakei 0,1 and to detect possible links between EPS yields and the activity of relevant enzymes. Methods and Results:,Fermentation experiments at different temperatures using either glucose or lactose were carried out. EPS production took place during the exponential growth phase. Low temperatures, applying glucose as carbohydrate source, resulted in the best bacterial growth, the highest amounts of EPS and the highest specific EPS production. Activities of 10 important enzymes involved in the EPS biosynthesis and the energy formation of Lact. sakei 0,1 were measured. The obtained results revealed that there is a clear link for some enzymes with EPS biosynthesis. It was also demonstrated clearly that the presence of rhamnose in the EPS building blocks is due to high activities of the enzymes involved in the rhamnose synthetic branch. Conclusions:,EPS production in Lact. sakei 0,1 is growth-associated and displays primary metabolite kinetics. Glucose as carbohydrate source and low temperatures enhance the EPS production. The enzymes involved in the biosynthesis of the activated sugar nucleotides play a major role in determining the monomeric composition of the synthesized EPS. Significance and Impact of the Study:,The proposed results contribute to a better understanding of the physiological factors influencing EPS production and the key enzymes involved in EPS biosynthesis by Lact. sakei. [source] Effect of galactose and glucose on the exopolysaccharide production and the activities of biosynthetic enzymes in Lactobacillus casei CRL 87JOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2001F. Mozzi Aims: The objective of this work was to study the influence of the sugar source on exopolysaccharide (EPS) production and the activities of the enzymes involved in the synthesis of sugar nucleotides in Lactobacillus casei CRL 87. The relationship between these enzymes and EPS formation was determined. Methods and Results: The concentration of EPS was estimated by the phenol/sulphuric acid method while the chemical composition of purified EPS was investigated using gas-liquid chromatography. Biosynthetic enzyme activities were determined spectrophotometrically by measuring the formation or disappearance of NAD(P)H at 340 nm. Polysaccharide production by Lb. casei CRL 87 was 1·7 times greater on galactose than on glucose. The isolated polymer was composed of rhamnose, glucose and galactose. The activities of uridine-diphosphate (UDP)-glucose-pyrophosphorylase, thymidine-diphosphate (dTDP)-glucose-pyrophosphorylase and the dTDP-rhamnose-synthetic enzyme system were higher in galactose-grown than in glucose-grown cells. When an EPS, mutant strain was used, galactokinase activity was not detected on galactose, this sugar not being available for the formation of sugar nucleotides for further EPS production. dTDP-glucose-pyrophosphorylase and dTDP-rhamnose-synthetic enzyme system activities were lower than the values found for the wild type strain. Conclusions: The carbon source present in the culture medium affects EPS production by Lb. casei CRL 87. The greater polymer synthesis by galactose-grown cells is correlated with the higher UDP-glucose-pyrophosphorylase, dTDP-glucose-pyrophosphorylase and dTDP-rhamnose-synthetic enzyme system activities. Initial sugar metabolism is also an important step for the synthesis of EPS precursors by this strain. Significance and Impact of the Study: Knowledge of the effect of the sugar source on EPS production and the activities of biosynthetic enzymes provides information about the mechanisms of regulation of the synthesis of EPS which can contribute to improving polymer production. [source] | ||||||||||