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The Biochemical Journal Feb 19681. Growth of Escherichia coli on glucosamine results in an induction of glucosamine 6-phosphate deaminase [2-amino-2-deoxy-d-glucose 6-phosphate ketol-isomerase...
1. Growth of Escherichia coli on glucosamine results in an induction of glucosamine 6-phosphate deaminase [2-amino-2-deoxy-d-glucose 6-phosphate ketol-isomerase (deaminating), EC 5.3.1.10] and a repression of glucosamine 6-phosphate synthetase (l-glutamine-d-fructose 6-phosphate aminotransferase, EC 2.6.1.16); glucose abolishes these control effects. 2. Growth of E. coli on N-acetylglucosamine results in an induction of N-acetylglucosamine 6-phosphate deacetylase and glucosamine 6-phosphate deaminase, and in a repression of glucosamine 6-phosphate synthetase; glucose diminishes these control effects. 3. The synthesis of amino sugar kinases (EC 2.7.1.8 and 2.7.1.9) is unaffected by growth on amino sugars. 4. Glucosamine 6-phosphate synthetase is inhibited by glucosamine 6-phosphate. 5. Mutants of E. coli that are unable to grow on N-acetylglucosamine have been isolated, and lack either N-acetylglucosamine 6-phosphate deacetylase (deacetylaseless) or glucosamine 6-phosphate deaminase (deaminaseless). Deacetylaseless mutants can grow on glucosamine but deaminaseless mutants cannot. 6. After growth on glucose, deacetylaseless mutants have a repressed glucosamine 6-phosphate synthetase and a super-induced glucosamine 6-phosphate deaminase; this may be related to an intracellular accumulation of acetylamino sugar that also occurs under these conditions. In one mutant the acetylamino sugar was shown to be partly as N-acetylglucosamine 6-phosphate. Deaminaseless mutants have no abnormal control effects after growth on glucose. 7. Addition of N-acetylglucosamine or glucosamine to cultures of a deaminaseless mutant caused inhibition of growth. Addition of N-acetylglucosamine to cultures of a deacetylaseless mutant caused lysis, and secondary mutants were isolated that did not lyse; most of these secondary mutants had lost glucosamine 6-phosphate deaminase and an uptake mechanism for N-acetylglucosamine. 8. Similar amounts of (14)C were incorporated from [1-(14)C]-glucosamine by cells of mutants and wild-type growing on broth. Cells of wild-type and a deaminaseless mutant incorporated (14)C from N-acetyl[1-(14)C]glucosamine more efficiently than from N[1-(14)C]-acetylglucosamine, incorporation from the latter being further decreased by acetate; cells of a deacetylaseless mutant showed a poor incorporation of both types of labelled N-acetylglucosamine.
Topics: Bacteriolysis; Carbon Isotopes; Cell Wall; Chromatography, Ion Exchange; Chromatography, Paper; Culture Media; Enzyme Induction; Enzyme Repression; Escherichia coli; Glucosamine; Glucose; Growth; Isomerases; Mutation; Phosphotransferases; Spectrum Analysis; Transaminases; Ultraviolet Rays
PubMed: 4866432
DOI: 10.1042/bj1060847 -
Biochimica Et Biophysica Acta Nov 2012Carbohydrates play a key role in the biological activity of numerous natural products. In many instances their biosynthesis requires radical mediated rearrangements,... (Review)
Review
Carbohydrates play a key role in the biological activity of numerous natural products. In many instances their biosynthesis requires radical mediated rearrangements, some of which are catalyzed by radical SAM enzymes. BtrN is one such enzyme responsible for the dehydrogenation of a secondary alcohol in the biosynthesis of 2-deoxystreptamine. DesII is another example that catalyzes a deamination reaction necessary for the net C4 deoxygenation of a glucose derivative en route to desosamine formation. BtrN and DesII represent the two most extensively characterized radical SAM enzymes involved in carbohydrate biosynthesis. In this review, we summarize the biosynthetic roles of these two enzymes, their mechanisms of catalysis, the questions that have arisen during these investigations and the insight they can offer for furthering our understanding of radical SAM enzymology. This article is part of a Special Issue entitled: Radical SAM enzymes and Radical Enzymology.
Topics: Amino Sugars; Bacterial Proteins; Biocatalysis; Biological Products; Free Radicals; Hexosamines; Iron-Sulfur Proteins; Mutation; Oxidoreductases; S-Adenosylmethionine; Stereoisomerism; Substrate Specificity
PubMed: 22172915
DOI: 10.1016/j.bbapap.2011.11.006 -
Journal of Bacteriology Jun 2016We have investigated the impact of growth on glucosamine (GlcN) and N-acetylglucosamine (GlcNAc) on cellular metabolism by quantifying glycolytic metabolites in...
UNLABELLED
We have investigated the impact of growth on glucosamine (GlcN) and N-acetylglucosamine (GlcNAc) on cellular metabolism by quantifying glycolytic metabolites in Escherichia coli Growth on GlcNAc increased intracellular pools of both GlcNAc6P and GlcN6P 10- to 20-fold compared to growth on glucose. Growth on GlcN produced a 100-fold increase in GlcN6P but only a slight increase in GlcNAc6P. Changes to the amounts of downstream glycolytic intermediates were minor compared to growth on glucose. The enzyme glucosamine-6P deaminase (NagB) is required for growth on both GlcN and GlcNAc. It is an allosteric enzyme in E. coli, displaying sigmoid kinetics with respect to its substrate, GlcN6P, and is allosterically activated by GlcNAc6P. The high concentration of GlcN6P, accompanied by the small increase in GlcNAc6P, drives E. coli NagB (NagBEc) into its high activity state, as observed during growth on GlcN (L. I. Álvarez-Añorve, I. Bustos-Jaimes, M. L. Calcagno, and J. Plumbridge, J Bacteriol 191:6401-6407, 2009, http://dx.doi.org/10.1128/JB.00633-09). The slight increase in GlcNAc6P during growth on GlcN is insufficient to displace NagC, the GlcNAc6P-responsive repressor of the nag genes, from its binding sites, so there is only a small increase in nagB expression. We replaced the gene for the allosteric NagBEc enzyme with that of the nonallosteric, B. subtilis homologue, NagBBs We detected no effects on growth rates or competitive fitness on glucose or the amino sugars, nor did we detect any effect on the concentrations of central metabolites, thus demonstrating the robustness of amino sugar metabolism and leaving open the question of the role of allostery in the regulation of NagB.
IMPORTANCE
Chitin, the polymer of N-acetylglucosamine, is an abundant biomaterial, and both glucosamine and N-acetylglucosamine are valuable nutrients for bacteria. The amino sugars are components of numerous essential macromolecules, including bacterial peptidoglycan and mammalian glycosaminoglycans. Controlling the biosynthetic and degradative pathways of amino sugar metabolism is important in all organisms to avoid loss of nitrogen and energy via a futile cycle of synthesis and breakdown. The enzyme glucosamine-6P deaminase (NagB) is central to this control, and N-acetylglucosamine-6P is the key signaling molecule regulating amino sugar utilization in Escherichia coli Here, we investigate how the metabolic status of the bacteria impacts on the activity of NagBEc and the N-acetylglucosamine-6P-sensitive transcriptional repressor, NagC.
Topics: Aldose-Ketose Isomerases; Amino Sugars; Enzyme Activation; Escherichia coli; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Mutation; Organisms, Genetically Modified; Repressor Proteins
PubMed: 27002132
DOI: 10.1128/JB.00870-15 -
Journal of Bacteriology Nov 2015The ability of bacteria to metabolize glucosamine (GlcN) and N-acetyl-d-glucosamine (GlcNAc) is considered important for persistent colonization of the oral cavity. In...
UNLABELLED
The ability of bacteria to metabolize glucosamine (GlcN) and N-acetyl-d-glucosamine (GlcNAc) is considered important for persistent colonization of the oral cavity. In the dental caries pathogen Streptococcus mutans, the NagR protein regulates the expression of glmS, which encodes a GlcN-6-P synthetase, and nagA (GlcNAc-6-P deacetylase) and nagB (GlcN-6-P deaminase), which are required for the catabolism of GlcNAc and GlcN. Two NagR-binding sites (dre) were identified in each of the promoter regions for nagB and glmS. Using promoter-reporter gene fusions, the role of each dre site was examined in the regulation of glmS and nagB promoter activities in cells grown with glucose, GlcNAc, or GlcN. A synergistic relationship between the two dre sites in the glmS promoter that required proper spacing was observed, but that was not the case for nagB. Binding of purified NagR to DNA fragments from both promoter regions, as well as to dre sites alone, was strongly influenced by particular sugar phosphates. Using a random mutagenesis approach that targeted the effector-binding domain of NagR, mutants that displayed aberrant regulation of both the glmS and nagAB genes were identified. Collectively, these findings provide evidence that NagR is essential for regulation of genes for both the synthesis and catabolism of GlcN and GlcNAc in S. mutans, and that NagR engages differently with the target promoter regions in response to specific metabolites interacting with the effector-binding domain of NagR.
IMPORTANCE
Glucosamine and N-acetylglucosamine are among the most abundant naturally occurring sugars on the planet, and they are catabolized by many bacterial species as sources of carbon and nitrogen. Representing a group called lactic acid bacteria (LAB), the human dental caries pathogen Streptococcus mutans is shown to differ from known paradigm organisms in that it possesses a GntR/HutC-type regulator, NagR, that is required for the regulation of both catabolism of GlcN and biosynthesis. Results reported here reveal a simple and elegant mechanism whereby NagR differentially regulates two opposing biological processes by surveying metabolic intermediates. This study provides insights that may contribute to the development of novel therapeutic tools to combat dental caries and other infectious diseases.
Topics: Amino Acid Sequence; Amino Sugars; Bacterial Proteins; Base Sequence; Gene Expression Regulation, Bacterial; Molecular Sequence Data; Streptococcus mutans
PubMed: 26324448
DOI: 10.1128/JB.00606-15 -
PloS One 2011Metabolic pathways for amino sugars (N-acetylglucosamine; GlcNAc and glucosamine; Gln) are essential and remain largely conserved in all three kingdoms of life, i.e.,...
Metabolic pathways for amino sugars (N-acetylglucosamine; GlcNAc and glucosamine; Gln) are essential and remain largely conserved in all three kingdoms of life, i.e., microbes, plants and animals. Upon uptake, in the cytoplasm these amino sugars undergo phosphorylation by phosphokinases and subsequently deacetylation by the enzyme N-acetylglucosamine 6-phosphate deacetylase (nagA) to yield glucosamine-6-phosphate and acetate, the first committed step for both GlcNAc assimilation and amino-sugar-nucleotides biosynthesis. Here we report the cloning of a DNA fragment encoding a partial nagA gene and its implications with regard to amino sugar metabolism in the cellulose producing bacterium Glucoacetobacter xylinus (formally known as Acetobacter xylinum). For this purpose, nagA was disrupted by inserting tetracycline resistant gene (nagA::tet(r); named as ΔnagA) via homologous recombination. When compared to glucose fed conditions, the UDP-GlcNAc synthesis and bacterial growth (due to lack of GlcNAc utilization) was completely inhibited in nagA mutants. Interestingly, that inhibition occured without compromising cellulose production efficiency and its molecular composition under GlcNAc fed conditions. We conclude that nagA plays an essential role for GlcNAc assimilation by G. xylinus thus is required for the growth and survival for the bacterium in presence of GlcNAc as carbon source. Additionally, G. xylinus appears to possess the same molecular machinery for UDP-GlcNAc biosynthesis from GlcNAc precursors as other related bacterial species.
Topics: Acetylglucosamine; Amidohydrolases; Amino Acid Sequence; Bacterial Proteins; Cloning, Molecular; Cytoplasm; Gluconacetobacter xylinus; Microbial Viability; Microscopy, Atomic Force; Molecular Sequence Data; Mutation; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Uridine Diphosphate Sugars
PubMed: 21655093
DOI: 10.1371/journal.pone.0018099 -
American Journal of Physiology. Renal... Mar 2015Galectin-3 activation is involved in the pathogenesis of renal damage and fibrogenesis. Limited data are available to suggest that galectin-3-targeted intervention is a...
Galectin-3 activation is involved in the pathogenesis of renal damage and fibrogenesis. Limited data are available to suggest that galectin-3-targeted intervention is a potential therapeutic candidate for the prevention of chronic kidney disease. Homozygous TGR(mREN)27 (REN2) rats develop severe high blood pressure (BP) and hypertensive end-organ damage, including nephropathy and heart failure. Male REN2 rats were treated with N-acetyllactosamine [galectin-3 inhibitor (Gal3i)] for 6 wk; untreated REN2 and Sprague-Dawley rats served as controls. We measured cardiac function with echocardiogram and invasive hemodynamics before termination. BP and proteinuria were measured at baseline and at 3 and 6 wk. Plasma creatinine was determined at 6 wk. Renal damage was assessed for focal glomerular sclerosis, glomerular desmin expression, glomerular and interstitial macrophages, kidney injury molecule-1 expression, and α-smooth muscle actin expression. Inflammatory cytokines and extracellular matrix proteinases were quantified by quantitative real-time PCR. Systolic BP was higher in control REN2 rats, with no effect of Gal3i treatment. Plasma creatinine and proteinuria were significantly increased in control REN2 rats; Gal3i treatment reduced both. Renal damage (focal glomerular sclerosis, desmin, interstitial macrophages, kidney injury molecule-1, α-smooth muscle actin, collagen type I, and collagen type III) was also improved by Gal3i. All inflammatory markers (CD68, IL-68, galectin-3, and monocyte chemoattractant protein-1) were elevated in control REN2 rats and attenuated by Gal3i. Markers of extracellular matrix turnover were marginally altered in untreated REN2 rats compared with Sprague-Dawley rats. In conclusion, galectin-3 inhibition attenuated hypertensive nephropathy, as indicated by reduced proteinuria, improved renal function, and decreased renal damage. Drugs binding to galectin-3 may be therapeutic candidates for the prevention of chronic kidney disease.
Topics: Amino Sugars; Animals; Drug Evaluation, Preclinical; Galectin 3; Hypertension; Kidney Diseases; Male; Rats, Sprague-Dawley
PubMed: 25503732
DOI: 10.1152/ajprenal.00461.2014 -
Molecules (Basel, Switzerland) Dec 2022Herein, we present a mild strategy for deprotecting cyclic sulfamidates via the Kukhtin-Ramirez reaction to access amino sugars. The method features the removal of the...
Herein, we present a mild strategy for deprotecting cyclic sulfamidates via the Kukhtin-Ramirez reaction to access amino sugars. The method features the removal of the sulfonic group of cyclic sulfamidates, which occurs through an N-H insertion reaction that implicates the Kukhtin-Ramirez adducts, followed by a base-promoted reductive N-S bond cleavage. The mild reaction conditions of the protocol enable the formation of amino alcohols including analogs that bear multiple functional groups.
Topics: Amino Sugars; Sulfones
PubMed: 36615376
DOI: 10.3390/molecules28010182 -
Bioorganic & Medicinal Chemistry Letters Oct 2015Two novel α-linked sialyltrisaccharide imidazolium-type probes (ITags) based on the structures of biologically relevant 6'-sialyllactosamine and 3'-sialyllactosamine...
Two novel α-linked sialyltrisaccharide imidazolium-type probes (ITags) based on the structures of biologically relevant 6'-sialyllactosamine and 3'-sialyllactosamine were efficiently and stereoselectively prepared using a chemo-enzymatic approach. The apparent kinetic parameters for the enzyme catalyzed transformations with α-2,3-sialyltransferase (α-2,3-ST) and α-2,6-sialyltransferase (α-2,6-ST) were measured by LC-MS using the ionic probes. This strategy demonstrates the suitability of the ITags to probe glycosyltransferase activity and their versatility in the preparation of sialylated epitopes for glycobiology research.
Topics: Amino Sugars; Carbohydrate Conformation; Imidazoles; Molecular Probes; Sialyltransferases
PubMed: 26318990
DOI: 10.1016/j.bmcl.2015.07.049 -
The Biochemical Journal Mar 1997This study presents a comparison of heparan sulphate chains isolated from various porcine and bovine tissues. 1H-NMR spectroscopy (500 MHz) was applied for structural... (Comparative Study)
Comparative Study
This study presents a comparison of heparan sulphate chains isolated from various porcine and bovine tissues. 1H-NMR spectroscopy (500 MHz) was applied for structural and compositional studies on intact heparan sulphate chains. After enzymic digestion of heparan sulphate using heparin lyase I (EC 4.2.2.7) II and III (EC 4.2.2.8), the compositions of unsaturated disaccharides obtained were determined by analytical capillary electrophoresis. Correlations between the N-sulphated glucosamine residues and O-sulphation and between iduronic acid content and total sulphation were discovered using the data obtained by NMR and disaccharide analysis. Heparan sulphate chains could be classified into two groups based on the sulphation degree and the iduronic acid content. Heparan sulphate chains with a high degree of sulphation possessed also a significant number of iduronic acid residues and were isolated exclusively from porcine brain, liver and kidney medulla. The presence and amount of N-unsubstituted glucosamine residues (GlcNp) was established in all of the heparan sulphates examined. The structural context in which this residue occurs was demonstrated to be: high sulphation domain --> 4)-beta-D-GlcAp-(1 --> 4)-alpha-D-GlcNp-(1 --> 4)-beta-D-GlcAp-(1 --> low sulphation domain (where GlcNp is 2-amino-2-deoxyglucopyranose, and GlcAp is glucopyranosyluronic acid), based on the isolation and characterization of a novel, heparin lyase III-derived, GlcNp containing tetrasaccharide and hexasaccharide. The results presented suggest that structural differences may play a role in important biological events controlled by heparan sulphate in different tissues.
Topics: Amino Sugars; Animals; Carbohydrate Sequence; Cattle; Disaccharides; Electrophoresis, Capillary; Glucosamine; Heparin; Heparitin Sulfate; Iduronic Acid; Magnetic Resonance Spectroscopy; Male; Molecular Sequence Data; Molecular Weight; Oligosaccharides; Species Specificity; Swine; Tissue Distribution
PubMed: 9065769
DOI: 10.1042/bj3220499 -
Cell Nov 2019Plasmodium gene functions in mosquito and liver stages remain poorly characterized due to limitations in the throughput of phenotyping at these stages. To fill this...
Plasmodium gene functions in mosquito and liver stages remain poorly characterized due to limitations in the throughput of phenotyping at these stages. To fill this gap, we followed more than 1,300 barcoded P. berghei mutants through the life cycle. We discover 461 genes required for efficient parasite transmission to mosquitoes through the liver stage and back into the bloodstream of mice. We analyze the screen in the context of genomic, transcriptomic, and metabolomic data by building a thermodynamic model of P. berghei liver-stage metabolism, which shows a major reprogramming of parasite metabolism to achieve rapid growth in the liver. We identify seven metabolic subsystems that become essential at the liver stages compared with asexual blood stages: type II fatty acid synthesis and elongation (FAE), tricarboxylic acid, amino sugar, heme, lipoate, and shikimate metabolism. Selected predictions from the model are individually validated in single mutants to provide future targets for drug development.
Topics: Alleles; Amino Sugars; Animals; Culicidae; Erythrocytes; Fatty Acid Synthases; Fatty Acids; Gene Knockout Techniques; Genome, Protozoan; Genotype; Life Cycle Stages; Liver; Models, Biological; Mutation; Parasites; Phenotype; Plasmodium berghei; Ploidies; Reproduction
PubMed: 31730853
DOI: 10.1016/j.cell.2019.10.030