Screening assay for inhibitors of a recombinant Streptococcus pneumoniae UDP-glucose pyrophosphorylase

The UDP-glucose pyrophosphorylase of Streptococcus pneumoniae (GalU_Spn) is absolutely required for the biosynthesis of capsular polysaccharide, the sine qua non virulence factor of pneumococcus. Since the eukaryotic enzymes are completely unrelated to their prokaryotic counterparts, we propose that the GalU enzyme is a critical target to fight the pneumococcal disease. A recombinant GalU_Spn was overexpressed and purified. An enzymatic assay that is rapid, sensitive and easy to perform was developed. This assay was appropriate for screening chemical libraries for searching GalU inhibitors. This work represents a fundamental step in the exploration of novel antipneumococcal drugs.     

UDP-glucose Pyrophosphorylase from Tubers of Jerusalem Artichoke (Helianthus tuberosus L.)

UDP-glucose pyrophosphorylase of Jerusalem artichoke tubers was purified 90-fold over the crude extract. The purified enzyme preparation absolutely required magnesium ions for activity. Cobalt ions were 60% as effective as magnesium ions; other divalent cations including manganese showed little or no effect. This enzyme had a pH optimum of 8.5 and a temperature optimum of 40°C. ATP and UDP inhibited the activity of this enzyme in both forward and backward directions. Km values for UDP-glucose, inorganic pyrophosphate, glucose-1-phosphate and UTP were determined to be 4.45 × 10^?4 M, 2.33 × 10^?4 M, 9.38 × 10^?4 M and 2.98 × 10^?4 M, respectively. These results are discussed in comparison with those of UDP-glucose pyrophosphorylases isolated from other plants.     

Structural basis for the reaction mechanism of UDP-glucose pyrophosphorylase

UDP-glucose pyrophosphorylases (UGPase; EC 2.7.7.9) catalyze the conversion of UTP and glucose-1-phosphate to UDP-glucose and pyrophosphate and vice versa. Prokaryotic UGPases are distinct from their eukaryotic counterparts and are considered appropriate targets for the development of novel antibacterial agents since their product, UDP-glucose, is indispensable for the biosynthesis of virulence factors such as lipopolysaccharides and capsular polysaccharides. In this study, the crystal structures of UGPase from Helicobacter pylori (HpUGPase) were determined in apo- and UDP-glucose/Mg²⁺-bound forms at 2.9 Å and 2.3 Å resolutions, respectively. HpUGPase is a homotetramer and its active site is located in a deep pocket of each subunit. Magnesium ion is coordinated by Asp130, two oxygen atoms of phosphoryl groups, and three water molecules with octahedral geometry. Isothermal titration calorimetry analyses demonstrated that Mg²⁺ ion plays a key role in the enzymatic activity of UGPase by enhancing the binding of UGPase to UTP or UDP-glucose, suggesting that this reaction is catalyzed by an ordered sequential Bi Bi mechanism. Furthermore, the crystal structure explains the specificity for uracil bases. The current structural study combined with functional analyses provides essential information for understanding the reaction mechanism of bacterial UGPases, as well as a platform for the development of novel antibacterial agents.     

Analysis of glucose 1-phosphate at the picomole level with [C]UTP and uridine 5′-diphosphoglucose pyrophosphorylase

A new sensitive method is described for glucose 1-phosphate analysis. The key reaction is the pyrophosphorolysis of UDP-glucose catalyzed by uridine 5′-diphosphoglucose pyrophosphorylase. The reaction product, [¹⁴C]UDP-glucose, is separated from [¹⁴C]UTP by adsorbing [¹⁴C]UTP selectively onto polyethyleneimine cellulose or by separating both labeled compounds on one-dimensional polyethyleneimine thin-layer chromatograms. The sensitivity of the method for glucose 1-phosphate analysis is 5 pmol. The method has been successfully employed to monitor the level of glucose 1-phosphate in early germination of wheat embryos.     

The galU gene of Xanthomonas campestris pv. campestris is involved in bacterial attachment,cell motility,polysaccharide synthesis,virulence, and tolerance to various stresses

Uridine triphosphate (UTP)-glucose-1-phosphate uridylyltransferase (GalU; EC 2.7.7.9) is an enzyme that catalyzes the formation of uridine diphosphate (UDP)-glucose from UTP and glucose-1-phosphate. GalU is involved in virulence in a number of animal-pathogenic bacteria since its product, UDP-glucose, is indispensable for the biosynthesis of virulence factors such as lipopolysaccharide and exopolysaccharide. However, its function in Xanthomonas campestris pv. campestris, the phytopathogen that causes black rot in cruciferous plants, is unclear. Here, we characterized a galU mutant of X. campestris pv. campestris and showed that the X. campestris pv. campestris galU mutant resulted in a reduction in virulence on the host cabbage. We also demonstrated that galU is involved in bacterial attachment, cell motility, and polysaccharide synthesis. Furthermore, the galU mutant showed increased sensitivity to various stress conditions including copper sulfate, hydrogen peroxide, and sodium dodecyl sulfate. In addition, mutation of galU impairs the expression of the flagellin gene fliC as well as the attachment-related genes xadA, fhaC, and yapH. In conclusion, our results indicate involvement of galU in the virulence factor production and pathogenicity in X. campestris pv. campestris, and a role for galU in stress tolerance of this crucifer pathogen.     

Biosynthèse de l'udpglucose par les microsomes des hépatocytes de ratBiosynthesis of UDPglucose by rat liver microsomes

Evidence is presented to show that all enzymes and all intermediary metabolites of a UDPglucose biosynthesis pathway are present in the microsomal membranes of rat liver. Glucose 6-phosphate, glucose 1-phosphate and UDPglucose are characterized by chromatography.The properties of phosphoglucomutase and UTP: D-Glucose-1-phosphate uridyltransferase are studied. The K_m values of phosphoglucomutase at pH 7.2 and 42°C were 0.26 · 10^?3 mM for glucose 1,6-diphosphate and 80 · 10^?3 mM for glucose 1-phosphate. The K_m values of UTP: D-glucose-1-phosphate uridyltransferase at pH 8.5 and 37°C were 220 · 10^?3 mM for UTP and 166 · 10^?3 mM for glucose 1-phosphate. These values are compared to the given values for enzymes from different species, and to those found for soluble enzymes. The significance of this membranous pathway is discussed.     

Molecular cloning of the Leishmania major UDP-glucose pyrophosphorylase, functional characterization, and ligand binding analyses using NMR spectroscopy

The dense glycocalyx surrounding the protozoan parasite Leishmania is an essential virulence factor. It protects the parasite from hostile environments in the sandfly vector and mammalian host and supports steps of development and invasion. Therefore, new therapeutic concepts concentrate on disturbing glycocalyx biosynthesis. Deletion of genes involved in the metabolism of galactose and mannose have been shown to drastically reduce Leishmania virulence. Here we report the identification of Leishmania major UDP-glucose pyrophosphorylase (UGP). UGP catalyzes the formation of UDP-glucose from glucose 1-phosphate and UTP. This activation step enables glucose to enter metabolic pathways and is crucial for the activation of galactose. UDP-galactose is made from UDP-glucose by nucleotide-donor transfer to galactose 1-phosphate or by epimerization of the glucose moiety. Isolated in a complementation cloning approach, the activity of L. major UGP was proven in vitro. Moreover, purified protein was used to investigate enzyme kinetics, quaternary organization, and binding of ligands. Whereas sequestration by oligomerization is a known regulatory mechanism for eukaryotic UGPs, the recombinant as well as native L. major UGP migrated as monomer in size exclusion chromatography and in accord with this showed simple Michaelis-Menten kinetics toward all substrates. In saturation transfer difference (STD)-NMR studies, we clearly demonstrated that the molecular geometry at position 4 of glucose is responsible for substrate specificity. Furthermore, the gamma-phosphate group of UTP is essential for binding and for induction of the open conformation, which then allows entry of glucose 1-phosphate. Our data provide the first direct proof for the ordered bi-bi mechanism suggested in earlier studies.     

The GalF protein of Escherichia coli is not a UDP-glucose pyrophosphorylase but interacts with the GalU protein possibly to regulate cellular levels of UDP-glucose

We report the functional characterization of the galF gene of strain VW187 ( Escherichia coli O7:K1), which encodes a polypeptide displaying structural features common to bacterial UDP-glucose pyrophosphorylases, including the E. coli GalU protein. These enzymes catalyse a reversible reaction converting UTP and glucose-1-phosphate into UDP-glucose and PP_i. We show that, although the GalF protein is expressed in vivo , GalF-expressing plasmids cannot complement the phenotype of a galU mutant and extracts from this mutant which only produces GalF are enzymatically inactive. In contrast, the presence of GalU and GalF proteins in the same cell-free extract caused a significant reduction in the rate of pyrophosphorolysis (conversion of UDP-glucose into glucose-1-phosphate) but no significant effect on the kinetics of synthesis of UDP-glucose. The presence of GalF also increased the thermal stability of the enzyme in vitro. The effect of GalF in the biochemical properties of the UDP-glucose pyrophosphorylase required the co-synthesis of GalF and GalU, suggesting that they could interact as components of the oligomeric enzyme. The physical interaction of GalU and GalF was demonstrated in vivo by the co-expression of both proteins as fusion products using a yeast two-hybrid system. Furthermore, using a pair of galF  ⁺/ galU ⁺ and galF/galU  ⁺ isogenic strains, we demonstrated that the presence of GalF is associated with an increased concentration of intracellular UDP-glucose as well as with an enhancement of the thermal stability of the UDP-glucose pyrophosphorylase in vivo . We propose that GalF is a non-catalytic subunit of the UDP-glucose pyrophosphorylase modulating the enzyme activity to increase the formation of UDP-glucose, and this function is important for bacterial adaptation to conditions of stress.     

Identification of α-<Emphasis Type="SmallCaps">d</Emphasis>-glucose-1-phosphate cytidylyltransferase involved in Ebosin biosynthesis of <Emphasis Type="BoldItalic">Streptomyces</Emphasis> sp. 139

Ebosin, a novel exopolysaccharide produced by Streptomyces sp. 139 has antagonist activity for IL-1R in vitro and remarkable anti-rheumatic arthritis activity in vivo. Its biosynthesis gene cluster (ste) has been identified. In this study, gene ste17 was expressed in Escherichia coli BL21 and the recombinant protein was purified. With CTP and α-d-glucose-1-phosphate as substrates, the recombinant Ste17 protein was found capable of catalyzing the production of CDP-d-glucose and pyrophosphate, demonstrating its identity as an α-d-glucose-1-phosphate–cytidylyltransferase (CDP-d-glucose synthase). To investigate the function of ste17 in Ebosin biosynthesis, the gene was disrupted with a double crossover via homologous recombination. The monosaccharide composition of exopolysaccharide (EPS) produced by the mutant Streptomyces sp. 139 (ste17 ⁻) was found significantly altered from that of Ebosin, with glucose becoming undetectable. This gene knockout also negatively affected the antagonist activity for IL-1R of EPS. These results indicate that the CDP-d-glucose synthase encoded by ste17 gene is involved in the formation of nucleotide sugar (CDP-d-glucose) as glucose precursor in Ebosin biosynthesis. Xiao-Qiang Qi and Qing-Li Sun contributed equally to this work.     

外源糖对水稻Ugp1基因表达调控研究

植物尿苷二磷酸葡萄糖焦磷酸化酶（UGPase）是蔗糖合成与降解途径的关键酶。本研究采用水稻叶片离体培养方法,结合Northern杂交技术,研究了外源糖对水稻Ugp1基因表达的影响。研究结果表明,蔗糖、葡萄糖、果糖、光照均能上调水稻Ugp1基因的表达,同时这种上调表达依赖于己糖激酶;果糖能上调水稻成熟叶片中Ugp1基因的表达,但并不影响苗期叶片中Ugp1基因的表达,具组织特异性;葡萄糖和果糖协同作用对Ugp1基因的诱导表达强于蔗糖,这种诱导除依赖于己糖激酶外,还存在其它未知的调控途径。水稻中存在UGPase的多种异构体,蔗糖及光照可诱导水稻Ugp1基因的上调表达,但对水稻UGPase的多种异构体形式并无影响。研究结果将有助于深入了解水稻Ugp1基因与糖信号途径互作调控网络。     

CugP Is a Novel Ubiquitous Non-GalU-Type Bacterial UDP-Glucose Pyrophosphorylase Found in Cyanobacteria

UDP-glucose pyrophosphorylase synthesizes UDP-glucose from UTP and glucose 1-phosphate and exists in almost all species. Most bacteria possess a GalU-type UDP-glucose pyrophosphorylase, whereas many cyanobacteria species do not. In certain cyanobacteria, UDP-glucose is used as a substrate for synthesis of exopolysaccharide cellulose in spite of the absence of GalU-type UDP-glucose pyrophosphorylase. Therefore, there should be an uncharacterized UDP-glucose pyrophosphorylase in cyanobacteria. Here, we show that all cyanobacteria possess a non-GalU-type bacterial UDP-glucose pyrophosphorylase, i.e., CugP, a novel family in the nucleotide triphosphate transferase superfamily. The expressed recombinant Synechocystis sp. strain PCC 6803 CugP had pyrophosphorylase activity that was highly specific for UTP and glucose 1-phosphate. The fact that the CugP gene cannot be deleted completely in Synechocystis sp. PCC 6803 suggests its central role as the substrate supplier for galactolipid synthesis. Galactolipids are major constituents of the photosynthetic thylakoid membrane and important for photosynthetic activity. Based on phylogenetic analysis, this CugP-type UDP-glucose pyrophosphorylase may have recently been horizontally transferred to certain noncyanobacteria.     

Effect of 2-deoxy-d-glucose on maintenance in culture of neonatal B cell of rat

Summary The effect of 2-deoxy-d-glucose on maintenance in culture of B cells of the neonatal rat was examined by supplementation of Medium 199 containing 5.5 mM glucose with 1 mM 2-deoxy-d-glucose. Islets maintained in medium with 5.5 mM glucose (basal medium) for 7 d underwent remarkable decreases in glucose sensitivity, and the levels of insulin in the medium dropped. By contrast, addition of 2-deoxy-d-glucose promoted a higher insulin content in medium and an increase in the glucose-induced insulin release and biosynthesis. Moreover, the addition of the deoxysugar caused a selective deletion of fibroblasts and prevented the deterioration of islet cells in basal medium, yielding clusters mostly consisting of islet cells at the end of culture.     

The cyclic-2,3-diphosphoglycerate fromMethanobacterium thermoautotrophicum is thed enantiomer

The cyclic pyrophosphate obtained fromMethanobacterium thermoautotrophicum was converted tosn-glycerol 3-phosphate by a stereospecific route. This conversion establishes the structure as cyclic-2,3-diphospho-d-glycerate. The same method was used to determine the cellular content of this metabolite under two conditions: batch culture in a medium containing 2 mM inorganic phosphate and continuous culture with 0.1 mM phosphate in the inflowing medium. The values found were 194±13 and 27.5±1.1 mol/g dry weight, respectively. Computer modeling indicated that the pyrophosphate group cannot adopt a staggered conformation.     

Purification and properties ofl-serine dehydratase fromLactobacillus fermentum ATCC 14931

l-Serine dehydratase fromLactobacillus fermentum was purified 100-fold. It was stabilized by the presence of 1 mM l-cysteine in 50 mM phosphate buffer. M_r=150,000 was determined by gel filtration. The enzyme consists of four apparently identical subunits (M_r=40,000) that were observed after treatment with sodium dodecyl sulfate. The apparent K_m forl-serine was 65 mM. Fe⁺⁺ was required for the enzymatic activity, and the apparent K_m value for this reaction was 0.55 mM. Maximum enzymatic activity was observed at 45°C and pH 8.0 in 50 mM phosphate buffer. At pH values different from the optimum, a positive cooperativity between substrate molecules was observed. The activation energy of the reaction was 11,400 and 22,800 cal × mol^–1 for temperature values more than and less than 35°C respectively. The purified enzyme showed a maximum absorption between 400 and 420 nm, indicating the presence of pyridoxal-5-phosphate (PLP) as a prosthetic group. The PLP concentration was 0.027 µmoles per milligram of protein. The data suggest that there is 1 mol of PLP for each protein subunit.     

Breeding of a 5-Fluorouridine-resistant Mutant with Increased Cellulose Production from Acetobacter xylinum subsp. nonacetoxidans

UDP-glucose (UDP-G), the direct precursor of cellulose, is known to be produced from UTP and glucose-1-phosphate. In an attempt to increase UTP biosynthesis, 5-fluorouridine (5-FUR: a pyrimidine analog)-resistant mutants were obtained using Acetobacter xylinum subsp. nonacetoxidans 757 as the parent strain. One of the 5-FUR-resistant mutants, FUR-35, showed about 40% higher cellulose productivion compared to the parent strain. Intracellular levels of UTP and UDP-G in FUR-35 was found to be higher than those in the parent strain. The carbamyl phosphate synthetase II (CPS) activity of FUR-35 was higher than that of the parent strain and the feedback inhibition of CPS by UTP in FUR-35 had been released compared with that in the parent strain. These results suggest that the increased cellulose production of FUR-35 was attributable to its higher of intracellular UDP-G level resulting from increased UTP biosynthesis.     

The cytosolic isoenzyme of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase in Spinacia oleracea and other higher plants: extreme substrate ambiguity and other properties

The cytosolic isoenzyme of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (DS-Co: EC 4.1.2.15) in Spinacia oleracea, Solanum tubersosum and many other higher plants was found to use a diversity of substrates. Diose (glycolaldehyde), triose (D-glyceraldehyde, L-glyceraldehyde and DL-glyceraldehyde 3-phosphate), tetrose (D-erythrose, L-erythrose, D-erythrose 4-phosphate, D-threose and L-threose), and pentose (D-ribose 5-phosphate and D-arabinose 5-phosphate) were utilized in combination with phosphoenolpyruvate (PEP) to make the corresponding 2-keto-3-deoxy sugar acids. Glyoxylate was also utilized by DS-Co. Glycoladehyde exhibited the highest reaction velocity when substrates were tested at 3 mM concentrations. Pentoses were poor substrates except when phsophorylated, an effect which is probably due to an increased fraction of the molecules being in the open-chain form. Little stereoselective discrimination exists since comparable velocities were demonstrated with the D and L isomers of glyceraldehyde, erythrose or threose. The enzyme is not a reversible aldolase since pyruvate failed to substitute for PEP. The use of D-erythrose 4-phsophate or glycolaldehyde resulted in K_m values of 1.95 mM and 8.60 mM, respectively. However, glycolaldehyde exhibited the largest V_maxK_m ratio, suggesting a greater catalytic efficiency for this substrate. Glycolaldehyde is an ideal substrate for inexpensive assays of DS-Co that are absolutely selective in the presence of two other plant enzymes which also utilize erythrose 4-phosphate and PEP. The spinach DS-Co enzymes required divalent metals for activity. The presence of 20 mM Mg²⁺, 1 mM Co²⁺ and 1 mM Mn²⁺ yielded relative activities of 100, 70 and 15, respectively. The pH optimum was 9.5 and temperature optimum for activity was 49°C. The molecular masses of DS-Co from spinach, tobacco and pea were all in the range of 400 kDa. The possible roles of DS-Co in biosynthesis of α-ketoglutarate and aromatic amino acids, in biosynthesis of components of cell wall and phytotoxin, and in acting as a sink for 2-and 3-carbon sugars are discussed.     

Control of glucuronidation during hypoxia. Limitation by UDP-glucose pyrophosphorylase.

The regulation of glucuronidation during hypoxia was studied in isolated hepatocytes by analysing the dependence of acetaminophen glucuronidation rate on the intracellular concentrations of UTP, glucose 1-phosphate, UDP-glucose and UDP-glucuronic acid. The steady-state concentrations of these metabolites in cells from fed and starved rats were altered by exposure to various hypoxic O2 concentrations and by adding exogenous glucose. Changes in glucuronidation rate under all conditions were explained in terms of the concentrations of the substrates for UDP-glucose pyrophosphorylase, i.e. UTP and glucose 1-phosphate. Steady-state rates for the UDP-glucose pyrophosphorylase reaction, calculated by using published kinetic constants and measured glucose 1-phosphate and UTP concentrations, were in agreement with the measured glucuronidation rates. Thus the UDP-glucose pyrophosphorylase reaction is the key regulatory site for drug glucuronidation during hypoxia. Control at this site indicates that glucuronidation in vivo may be generally depressed in pathological conditions involving hypoxia and energy (calorie) malnutrition.     

Microphotometric determination of enzymes in brain sections

Summary A histochemical procedure was established for the microphotometric determination of hexokinase (HK) in sections of the rat hippocampus, which served as an exemplary brain region. For this quantitative procedure, slides were coated with glucose 6-phosphate dehydrogenase (G6PDH) as an auxiliary enzyme and sections were mounted onto this enzyme film. The sections were then incubated with the following adapted incubation medium: 5 mM d-glucose, 1.5 mM NADP, 7.5 mM ATP, 4 mM nitroblue tetrazolium chloride, 10 mM NaN₃, 10 mM MgCl₂, 0.25 mM phenazine methosulfate, 1 U/ml G6PDH, 22% polyvinyl alcohol in 0.05 M Hepes buffer; the final pH was 7.5. A linear response of the reaction was observed in the initial 10 min of reaction (kinetic and end-point measurements). The relationship between HK activity and section thickness was linear up to 5 m. The need for such thin sections is discussed in relation to the limited penetration of the auxiliary enzyme into the section. It is concluded that the quantitative demonstration of HK in brain sections could be a valuable tool for studying the local metabolic entrance of glucose in the glycolytic pathway.Supported by the Deutsche Forschungsgemeinschaft (Ku 541/2-1, 2-2)     

Genetic transformation using Agrobacterium rhizogenes

UDP-glucose pyrophosphorylase (EC 2.7.7.9) has been highly purified from the plant fraction of soybean ( Glycine max L. Merr. cv Williams) nodules. The purified enzyme gave a single polypeptide band following sodium docecyl sulphate polyacryla-mide gel electrophoresis, but was resolved into three bands of activity in non-denaturing gels. The enzyme appeared to be a monomer of molecular weight between 30 and 40 kDa. UDP-glucose pyrophosphorylase had optimum activity at pH 8.5 and displayed typical hyperbolic kinetics. The enzyme had a requirement for divalent metal ions, and was highly specific for the substrates pyrophosphate and UDP-glucose in the pyrophosphorolysis direction, and glucose-1-phosphate and UTP in the direction of UDP-glucose synthesis. The K_m values were 0.19 m M and 0.07 m M for pyrophosphate and UDP-glucose, respectively, and 0.23 m M and 0.11 m M for glucose-1-phosphate and UTP. The maximum velocity in the pyrophosphorolysis direction was almost double that for the reverse reaction. UDP-glucose pyrophosphorylase did not appear to be subject to a high degree of fine control, and activity in vivo may be regulated mainly by the availability of the substrates.     

Purification and characterization of benzoyl-CoA ligase from a syntrophic,benzoate-degrading,anaerobic mixed culture

Summary The benzoyl-CoA ligase from an anaerobic syntrophic culture was purified to homogeneity. It had a molecular mass of around 420 kDa and consisted of seven or eight subunits of 58 kDa. The temperature optimum was 37–40° C, the optimum pH around 8.0 and optimal activity required 50–100 mM TRIS-HCI buffer, pH 8.0 and 3–7 mM MgCl₂; MgCl₂ in excess of 10 mM was inhibitory. The activation energy for benzoate was 11.3 kcal/mol. Although growth occured only with benzoate as a carbon source, the benzoyl-coenzyme A (CoA) ligase formed benzoyl-CoA esters with benzoate, 2-, 3- and 4-fluorobenzoate, picolinate, nicotinate and isonicotinate. Acetate was activated to acetyl-CoA by an acetyl-CoA synthetase. The K _m values for benzoate, 2-, 3- and 4-fluorobenzoate were 0.04, 0.28, 1.48 and 0.32 mM, the V _max values 1.05, 1.0, 0.7 and 0.98 units (U)/mg, respectively. For reduced CoA (CoA-SH) a K _m of 0.17 mM and a V _max of 1.05 U/mg and for ATP a K _m of 0.16 mM and a V _max of 1.08 U/mg was determined. Benzoate activation was inhibited by more than 6 mM ATP, presumably by pyrophosphate generation from ATP. The inhibition constant (K _i) for pyrophosphate was 5.7 mM. No homology of the N-terminal amino acid sequence with that of a 2-aminobenzoyl-CoA ligase of a denitrifying Pseudomonas sp. was found. Correspondence to: J. Winter