Show legend Show as text

Structure type: monomer
Contained glycoepitopes: IEDB_137353,IEDB_140947,IEDB_2189047

• Article ID: 400
  Valvano MA "Biosynthesis and genetics of ADP-heptose" - Journal of Endotoxin Research (1999) 90-95
  

  Glycero-manno-heptose is a common component in the lipopolysaccharide (LPS) of many Gramnegative bacteria. Mutants deficient in the synthesis of glycero-manno-heptose are highly sensitive to hydrophobic compounds, and display reduced virulence, making these genes and their products potential targets for developing novel antimicrobials. To date, the biosynthesis of the heptosyl precursors for the inner core oligosaccharide of the LPS molecule is not completely characterized. In this work, the genes and enzyme functions involved in the various steps of the biosynthesis of ADP-Lglycero-D-manno-heptose are discussed, especially those involved in the intermediate steps.

  biosynthesis, genetic, genetics

  Publication DOI: 10.1177/09680519990050010901
  Journal NLM ID: 9433350
  Publisher: Maney Publishing
  Institutions: Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada
  Methods: genetic methods
  
   
  
  Haemophilus influenzae
  CSDB ID 8393 (all data & tools)

Show legend Show as text

Structure type: oligomer
Contained glycoepitopes: IEDB_137353,IEDB_140947

• Article ID: 661
  Gronow S, Oertelt C, Ervelä E, Zamyatina A, Kosma P, Skurnik M, Holst O "Characterization of the physiological substrate for lipopolysaccharide heptosyltransferases I and II" - Journal of Endotoxin Research 7(4) (2001) 263-270
  

  L-Glycero-D-manno-heptopyranose is a characteristic compound of many lipopolysaccharide (LPS) core structures of Gram-negative bacteria. In Escherichia coli two heptosyltransferases, namely WaaC and WaaF, are known to transfer L-glycero-D-manno-heptopyranose to Re-LPS and Rd(2)-LPS, respectively. It had been proposed that both reactions involve ADPL-glycero-D-manno-heptose as a sugar donor; however, the structure of this nucleotide sugar had never been completely elucidated. In the present study, ADPL-glycero-D-manno-heptose was isolated from a heptosyltransferase-deficient E. coli mutant, and its structure was determined by nuclear magnetic resonance spectroscopy and matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry as ADPL-glycero-β-D-manno-heptopyranose. This compound represented the sole constituent of the bacterial extract that was accepted as a sugar donor by heptosyltransferases I and II in vitro.

  Lipopolysaccharide, synthesis, heptosyltransferase, glycosyl transferases, heptoses, nucleotides

  NCBI PubMed ID: 11717579
  Journal NLM ID: 9433350
  Publisher: Maney Publishing
  Correspondence: oholst@fz-borstel.de
  Institutions: Medical and Biochemical Microbiology and Analytical Biochemistry, Research Center Borstel, Germany, Department of Medical Biochemistry, University of Turku, Finland, Department of Chemistry, University of Agricultural Sciences, Vienna, Austria
  Methods: NMR-2D, NMR, HPAEC, MALDI-TOF MS
  
   
  
  Escherichia coli WBB06
  CSDB ID 445 (all data & tools)

Show legend Show as text

Structure type: monomer
Trivial name: ADP-β-L-glycero-D-manno-heptose, ADP-L-glycero-β-D-manno-heptose
Contained glycoepitopes: IEDB_137353,IEDB_140947

• Article ID: 752
  Kneidinger B, Graninger M, Puchberger M, Kosma P, Messner P "Biosynthesis of nucleotide-activated D-glycero-D-manno-heptose" - Journal of Biological Chemistry 276(24) (2001) 20935-20944
  

  The glycan chain repeats of the S-layer glycoprotein of Aneurinibacillus thermoaerophilus DSM 10155 contain D-glycero-D-mannoheptose, which has also been described as constituent of lipopolysaccharide cores of Gram-negative bacteria. The four genes required for biosynthesis of the nucleotide-activated form GDP-D-glycero-D-mannoheptose were cloned, sequenced, and overexpressed in Escherichia coli, and the corresponding enzymes GmhA, GmhB, GmhC, and GmhD were purified to homogeneity. The isomerase GmhA catalyzed the conversion of D-sedoheptulose 7-phosphate to D-glycero-D-manno-heptose 7-phosphate, and the phosphokinase GmhB added a phosphate group to form D-glycero-D-manno-heptose 1,7-bisphosphate. The phosphatase GmhC removed the phosphate in the C-7 position, and the intermediate D-glycero-α-D-manno-heptose 1-phosphate was eventually activated with GTP by the pyrophosphorylase GmhD to yield the final product GDP-D-glycero-α-D-manno-heptose. The intermediate and end products were analyzed by high performance liquid chromatography. Nuclear magnetic resonance spectroscopy was used to confirm the structure of these substances. This is the first report of the biosynthesis of GDP-D-glycero-α-D-manno-heptose in Gram-positive organisms. In addition, we propose a pathway for biosynthesis of the nucleotide-activated form of L-glycero-L-manno-heptose

  Lipopolysaccharide, biosynthesis, L-glycero-D-manno-heptose, structure, core, heptose, intermediate, Bacterial Proteins, chemistry, gene, Bacterial, genetics, metabolism, structural, Support, Non-U.S.Gov't, chain, group, molecular, form, Escherichia, Escherichia coli, cloning, phosphate, high, lipopolysaccharide core, Molecular Sequence Data, Genes, Restriction Mapping, bacteria, glycan, position, Magnetic Resonance Spectroscopy, nuclear, nuclear magnetic resonance, nuclear magnetic resonance spectroscopy, resonance, spectroscopy, D-glycero-D-manno-heptose, Gram-negative bacteria, enzyme, gram negative bacteria, Gram-negative, homogeneity, purified, Carbohydrate Conformation, chromatography, Enzymes, pathway, cloned, Base Sequence, operon, amino acid, glycoproteins, Bacillus, conversion, activated, liquid chromatography, Gram-positive, S-layer, amino acid sequence, glycoprotein, S layer, heptoses, Sequence Alignment, Bacillaceae, High Pressure Liquid, DNA Primers, Guanosine Diphosphate Sugars, Membrane Glycoproteins, phosphatase, Phosphoric Monoester Hydrolases, Recombinant Proteins, Sequence Homology

  NCBI PubMed ID: 11279237
  Journal NLM ID: 2985121R
  Publisher: Baltimore, MD: American Society for Biochemistry and Molecular Biology
  Institutions: Zentrum fur Ultrastrukturforschung und Ludwig Boltzmann-Institut fur Molekulare Nanotechnologie, Universitat fur Bodenkultur Wien, Austria
  
   
  
  Escherichia coli
  CSDB ID 899 (all data & tools)
• Article ID: 3366
  Morrison JP, Tanner ME "A two-base mechanism for Escherichia coli ADP-L-glycero-D-manno-heptose 6-epimerase" - Biochemistry 46(12) (2007) 3916-3924
  

  ADP-L-glycero-D-manno-heptose 6-epimerase (HldD or AGME, formerly RfaD) catalyzes the inversion of configuration at C-6" of the heptose moiety of ADP-D-glycero-D-manno-heptose and ADP-L-glycero-D-manno-heptose. The epimerase HldD operates in the biosynthetic pathway of L-glycero-D-manno-heptose, which is a conserved sugar in the core region of lipopolysaccharide (LPS) of Gram-negative bacteria. Previous studies support a mechanism in which HldD uses its tightly bound NADP+ cofactor to oxidize directly at C-6", generating a ketone intermediate. A reduction of the ketone from the opposite face then occurs, generating the epimeric product. How the epimerase is able access both faces of the ketone intermediate with correct alignment of the three required components, NADPH, the ketone carbonyl, and a catalytic acid/base residue, is addressed here. It is proposed that the epimerase active site contains two catalytic pockets, each of which bears a catalytic acid/base residue that facilitates reduction of the C-6" ketone but leads to a distinct epimeric product. The ketone carbonyl may access either pocket via rotation about the C-5"-C-6" bond of the sugar nucleotide and in doing so presents opposing faces to the bound cofactor. Evidence in support of the two-base mechanism is found in studies of two single mutants of the Escherichia coli K-12 epimerase, Y140F and K178M, both of which have severely compromised epimerase activities that are more than 3 orders of magnitude lower than that of the wild type. The catalytic competency of these two mutants in promoting redox chemistry is demonstrated with an alternate catalytic activity that requires only one catalytic base: dismutation of a C-6" aldehyde substrate analogue (ADP-β-D-manno-hexodialdose) to an acid and an alcohol (ADP-β-d-mannuronic acid and ADP-β-D-mannose). This study identifies the two catalytic bases as tyrosine 140 and lysine 178. A one-step enzymatic conversion of mannose into ADP-β-mannose is also described and used to make C-6"-substituted derivatives of this sugar nucleotide

  Lipopolysaccharide, biosynthesis, L-glycero-D-manno-heptose, epimerase, sugar nucleotide, Escherichia coli K12

  NCBI PubMed ID: 17316025
  Journal NLM ID: 0370623
  Publisher: American Chemical Society
  Correspondence: mtanner@chem.ubc.ca
  Institutions: Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1
  Methods: 1H NMR, 31P NMR, ESI-MS, chemical methods, genetic methods
  
   
  
  Escherichia coli K12 W3110
  CSDB ID 21501 (all data & tools)
• Article ID: 3596
  Desroy N, Moreau F, Briet S, Fralliec GL, Floquet S, Durant L, Vongsouthi V, Gerusz V, Denis A, Escaich S "Towards Gram-negative antivirulence drugs: New inhibitors of HldE kinase" - Bioorganic and Medicinal Chemistry 17(3) (2008) 1276-1289
  

  Gram-negative bacteria lacking heptoses in their lipopolysaccharide (LPS) display attenuated virulence and increased sensitivity to human serum and to some antibiotics. Thus inhibition of bacterial heptose synthesis represents an attractive target for the development of new antibacterial agents. HldE is a bifunctional enzyme involved in the synthesis of bacterial heptoses. Development of a biochemical assay suitable for high-throughput screening allowed the discovery of inhibitors 1 and 2 of HldE kinase. Study of the structure-activity relationship of this series of inhibitors led to highly potent compounds.

  Lipopolysaccharide, Escherichia coli, antibacterial, antivirulence drugs, membrane permeability, HldE, RfaE

  NCBI PubMed ID: 19124251
  Journal NLM ID: 9413298
  Publisher: Elsevier
  Correspondence: N. Desroy
  Institutions: MUTABILIS SA, 102 Avenue Gaston Roussel, 93230 Romainville, France
  Methods: 1H NMR, ESI-MS, biochemical methods
  
   
  
  Escherichia coli
  CSDB ID 23513 (all data & tools)

Show legend Show as text

Structure type: monomer
Trivial name: ADP-β-D-glycero-D-manno-heptose
Contained glycoepitopes: IEDB_137353,IEDB_140947

• Article ID: 752
  Kneidinger B, Graninger M, Puchberger M, Kosma P, Messner P "Biosynthesis of nucleotide-activated D-glycero-D-manno-heptose" - Journal of Biological Chemistry 276(24) (2001) 20935-20944
  

  The glycan chain repeats of the S-layer glycoprotein of Aneurinibacillus thermoaerophilus DSM 10155 contain D-glycero-D-mannoheptose, which has also been described as constituent of lipopolysaccharide cores of Gram-negative bacteria. The four genes required for biosynthesis of the nucleotide-activated form GDP-D-glycero-D-mannoheptose were cloned, sequenced, and overexpressed in Escherichia coli, and the corresponding enzymes GmhA, GmhB, GmhC, and GmhD were purified to homogeneity. The isomerase GmhA catalyzed the conversion of D-sedoheptulose 7-phosphate to D-glycero-D-manno-heptose 7-phosphate, and the phosphokinase GmhB added a phosphate group to form D-glycero-D-manno-heptose 1,7-bisphosphate. The phosphatase GmhC removed the phosphate in the C-7 position, and the intermediate D-glycero-α-D-manno-heptose 1-phosphate was eventually activated with GTP by the pyrophosphorylase GmhD to yield the final product GDP-D-glycero-α-D-manno-heptose. The intermediate and end products were analyzed by high performance liquid chromatography. Nuclear magnetic resonance spectroscopy was used to confirm the structure of these substances. This is the first report of the biosynthesis of GDP-D-glycero-α-D-manno-heptose in Gram-positive organisms. In addition, we propose a pathway for biosynthesis of the nucleotide-activated form of L-glycero-L-manno-heptose

  Lipopolysaccharide, biosynthesis, L-glycero-D-manno-heptose, structure, core, heptose, intermediate, Bacterial Proteins, chemistry, gene, Bacterial, genetics, metabolism, structural, Support, Non-U.S.Gov't, chain, group, molecular, form, Escherichia, Escherichia coli, cloning, phosphate, high, lipopolysaccharide core, Molecular Sequence Data, Genes, Restriction Mapping, bacteria, glycan, position, Magnetic Resonance Spectroscopy, nuclear, nuclear magnetic resonance, nuclear magnetic resonance spectroscopy, resonance, spectroscopy, D-glycero-D-manno-heptose, Gram-negative bacteria, enzyme, gram negative bacteria, Gram-negative, homogeneity, purified, Carbohydrate Conformation, chromatography, Enzymes, pathway, cloned, Base Sequence, operon, amino acid, glycoproteins, Bacillus, conversion, activated, liquid chromatography, Gram-positive, S-layer, amino acid sequence, glycoprotein, S layer, heptoses, Sequence Alignment, Bacillaceae, High Pressure Liquid, DNA Primers, Guanosine Diphosphate Sugars, Membrane Glycoproteins, phosphatase, Phosphoric Monoester Hydrolases, Recombinant Proteins, Sequence Homology

  NCBI PubMed ID: 11279237
  Journal NLM ID: 2985121R
  Publisher: Baltimore, MD: American Society for Biochemistry and Molecular Biology
  Institutions: Zentrum fur Ultrastrukturforschung und Ludwig Boltzmann-Institut fur Molekulare Nanotechnologie, Universitat fur Bodenkultur Wien, Austria
  
   
  
  Escherichia coli
  CSDB ID 944 (all data & tools)
• Article ID: 753
  Kneidinger B, Marolda C, Graninger M, Zamyatina A, McArthur F, Kosma P, Valvano MA, Messner P "Biosynthesis pathway of ADP-L-glycero-b-D-manno-heptose in Escherichia coli" - Journal of Bacteriology 184(2) (2002) 363-369
  

  The steps involved in the biosynthesis of the ADP-L-glycero-β-D-manno-heptose (ADP-L-β-D-heptose) precursor of the inner core lipopolysaccharide (LPS) have not been completely elucidated. In this work, we have purified the enzymes involved in catalyzing the intermediate steps leading to the synthesis of ADP-D-β-D-heptose and have biochemically characterized the reaction products by high- performance anion-exchange chromatography. We have also constructed a deletion in a novel gene, gmhB (formerly yaeD), which results in the formation of an altered LPS core. This mutation confirms that the GmhB protein is required for the formation of ADP-D-β-D-heptose. Our results demonstrate that the synthesis of ADP-D-β-D-heptose in Escherichia coli requires three proteins, GmhA (sedoheptulose 7- phosphate isomerase), HldE (bifunctional D-β-D-heptose 7-phosphate kinase/D-β-D-heptose 1-phosphate adenylyltransferase), and GmhB (D,D- heptose 1,7-bisphosphate phosphatase), as well as ATP and the ketose phosphate precursor sedoheptulose 7-phosphate. A previously characterized epimerase, formerly named WaaD (RfaD) and now renamed HldD, completes the pathway to form the ADP-L-β-D-heptose precursor utilized in the assembly of inner core LPS

  Lipopolysaccharide, biosynthesis, synthesis, lipopolysaccharides, LPS, core, heptose, intermediate, gene, genetics, metabolism, phenotype, Support, Non-U.S.Gov't, form, Escherichia, Escherichia coli, phosphate, high, protein, assembly, epimerase, precursor, inner core, enzyme, mutation, formation, purified, chromatography, classification, Enzymes, pathway, gene expression, proteins, reaction, rfaD, enzymology, LPS core, phosphatase, Phosphoric Monoester Hydrolases, adenosine diphosphate sugars, anion-exchange, anion-exchange chromatography, ATP, Isomerases, ketose, Multienzyme Complexes, Nucleotidyltransferases, Phosphoprotein Phosphatase, Phosphotransferases (Alcohol Group Acceptor), Protein Kinases, Racemases and Epimerases, sedoheptulose, terminology

  NCBI PubMed ID: 11751812
  Journal NLM ID: 2985120R
  Publisher: American Society for Microbiology
  Institutions: Zentrum fur Ultrastrukturforschung und Ludwig Boltzmann-Institut fur Molekulare Nanotechnologie, A-1180 Vienna, Austria
  
   
  
  Escherichia coli K12
  CSDB ID 941 (all data & tools)
• Article ID: 3366
  Morrison JP, Tanner ME "A two-base mechanism for Escherichia coli ADP-L-glycero-D-manno-heptose 6-epimerase" - Biochemistry 46(12) (2007) 3916-3924
  

  ADP-L-glycero-D-manno-heptose 6-epimerase (HldD or AGME, formerly RfaD) catalyzes the inversion of configuration at C-6" of the heptose moiety of ADP-D-glycero-D-manno-heptose and ADP-L-glycero-D-manno-heptose. The epimerase HldD operates in the biosynthetic pathway of L-glycero-D-manno-heptose, which is a conserved sugar in the core region of lipopolysaccharide (LPS) of Gram-negative bacteria. Previous studies support a mechanism in which HldD uses its tightly bound NADP+ cofactor to oxidize directly at C-6", generating a ketone intermediate. A reduction of the ketone from the opposite face then occurs, generating the epimeric product. How the epimerase is able access both faces of the ketone intermediate with correct alignment of the three required components, NADPH, the ketone carbonyl, and a catalytic acid/base residue, is addressed here. It is proposed that the epimerase active site contains two catalytic pockets, each of which bears a catalytic acid/base residue that facilitates reduction of the C-6" ketone but leads to a distinct epimeric product. The ketone carbonyl may access either pocket via rotation about the C-5"-C-6" bond of the sugar nucleotide and in doing so presents opposing faces to the bound cofactor. Evidence in support of the two-base mechanism is found in studies of two single mutants of the Escherichia coli K-12 epimerase, Y140F and K178M, both of which have severely compromised epimerase activities that are more than 3 orders of magnitude lower than that of the wild type. The catalytic competency of these two mutants in promoting redox chemistry is demonstrated with an alternate catalytic activity that requires only one catalytic base: dismutation of a C-6" aldehyde substrate analogue (ADP-β-D-manno-hexodialdose) to an acid and an alcohol (ADP-β-d-mannuronic acid and ADP-β-D-mannose). This study identifies the two catalytic bases as tyrosine 140 and lysine 178. A one-step enzymatic conversion of mannose into ADP-β-mannose is also described and used to make C-6"-substituted derivatives of this sugar nucleotide

  Lipopolysaccharide, biosynthesis, L-glycero-D-manno-heptose, epimerase, sugar nucleotide, Escherichia coli K12

  NCBI PubMed ID: 17316025
  Journal NLM ID: 0370623
  Publisher: American Chemical Society
  Correspondence: mtanner@chem.ubc.ca
  Institutions: Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1
  Methods: 1H NMR, 31P NMR, ESI-MS, chemical methods, genetic methods
  
   
  
  Escherichia coli K12 W3110
  CSDB ID 21867 (all data & tools)

Show legend Show as text

Structure type: monomer
Trivial name: component of core oligosaccharide
Contained glycoepitopes: IEDB_137353,IEDB_140947

• Article ID: 753
  Kneidinger B, Marolda C, Graninger M, Zamyatina A, McArthur F, Kosma P, Valvano MA, Messner P "Biosynthesis pathway of ADP-L-glycero-b-D-manno-heptose in Escherichia coli" - Journal of Bacteriology 184(2) (2002) 363-369
  

  The steps involved in the biosynthesis of the ADP-L-glycero-β-D-manno-heptose (ADP-L-β-D-heptose) precursor of the inner core lipopolysaccharide (LPS) have not been completely elucidated. In this work, we have purified the enzymes involved in catalyzing the intermediate steps leading to the synthesis of ADP-D-β-D-heptose and have biochemically characterized the reaction products by high- performance anion-exchange chromatography. We have also constructed a deletion in a novel gene, gmhB (formerly yaeD), which results in the formation of an altered LPS core. This mutation confirms that the GmhB protein is required for the formation of ADP-D-β-D-heptose. Our results demonstrate that the synthesis of ADP-D-β-D-heptose in Escherichia coli requires three proteins, GmhA (sedoheptulose 7- phosphate isomerase), HldE (bifunctional D-β-D-heptose 7-phosphate kinase/D-β-D-heptose 1-phosphate adenylyltransferase), and GmhB (D,D- heptose 1,7-bisphosphate phosphatase), as well as ATP and the ketose phosphate precursor sedoheptulose 7-phosphate. A previously characterized epimerase, formerly named WaaD (RfaD) and now renamed HldD, completes the pathway to form the ADP-L-β-D-heptose precursor utilized in the assembly of inner core LPS

  Lipopolysaccharide, biosynthesis, synthesis, lipopolysaccharides, LPS, core, heptose, intermediate, gene, genetics, metabolism, phenotype, Support, Non-U.S.Gov't, form, Escherichia, Escherichia coli, phosphate, high, protein, assembly, epimerase, precursor, inner core, enzyme, mutation, formation, purified, chromatography, classification, Enzymes, pathway, gene expression, proteins, reaction, rfaD, enzymology, LPS core, phosphatase, Phosphoric Monoester Hydrolases, adenosine diphosphate sugars, anion-exchange, anion-exchange chromatography, ATP, Isomerases, ketose, Multienzyme Complexes, Nucleotidyltransferases, Phosphoprotein Phosphatase, Phosphotransferases (Alcohol Group Acceptor), Protein Kinases, Racemases and Epimerases, sedoheptulose, terminology

  NCBI PubMed ID: 11751812
  Journal NLM ID: 2985120R
  Publisher: American Society for Microbiology
  Institutions: Zentrum fur Ultrastrukturforschung und Ludwig Boltzmann-Institut fur Molekulare Nanotechnologie, A-1180 Vienna, Austria
  
   
  
  Escherichia coli
  CSDB ID 940 (all data & tools)
• Article ID: 3248
  Grizot S, Salem M, Vongsouthi V, Durand L, Moreau F, Dohi H, Vincent S, Escaich S, Ducruix A "Structure of the Escherichia coli Heptosyltransferase WaaC: Binary Complexes with ADP AND ADP-2-deoxy-2-fluoro Heptose" - Journal of Molecular Biology 363 (2006) 383-394
  

  Lipopolysaccharides constitute the outer leaflet of the outer membrane of Gram-negative bacteria and are therefore essential for cell growth and viability. The heptosyltransferase WaaC is a glycosyltransferase (GT) involved in the synthesis of the inner core region of LPS. It catalyzes the addition of the first l-glycero-d-manno-heptose (heptose) molecule to one 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) residue of the Kdo(2)-lipid A molecule. Heptose is an essential component of the LPS core domain; its absence results in a truncated lipopolysaccharide associated with the deep-rough phenotype causing a greater susceptibility to antibiotic and an attenuated virulence for pathogenic Gram-negative bacteria. Thus, WaaC represents a promising target in antibacterial drug design. Here, we report the structure of WaaC from the Escherichia coli pathogenic strain RS218 alone at 1.9 A resolution, and in complex with either ADP or the non-cleavable analog ADP-2-deoxy-2-fluoro-heptose of the sugar donor at 2.4 A resolution. WaaC adopts the GT-B fold in two domains, characteristic of one glycosyltransferase structural superfamily. The comparison of the three different structures shows that WaaC does not undergo a domain rotation, characteristic of the GT-B family, upon substrate binding, but allows the substrate analog and the reaction product to adopt remarkably distinct conformations inside the active site. In addition, both binary complexes offer a close view of the donor subsite and, together with results from site-directed mutagenesis studies, provide evidence for a model of the catalytic mechanism.

  Lipopolysaccharide, heptose, crystal structure, glycosyltransferase

  NCBI PubMed ID: 16963083
  Journal NLM ID: 2985088R
  Publisher: Elsevier
  Correspondence: arnaud.ducruix@univ-paris5.fr
  Institutions: Laboratoire de Cristallographie et RMN Biologiques, UMR 8015 CNRS, Universite Paris Descartes, Faculte de Pharmacie, 4, Avenue de lObservatoire, F-75270 Paris cedex 06, France, Mutabilis, 102, route de Noisy, F-93230 Romainville France, FUNDP Faculte des Sciences, Laboratoire de Chimie Bio-Organique, Rue de Bruxelles, 61, B-5000 Namur - Belgium, Ecole Normale Superieure, Departement de Chimie, UMR 8642 du CNRS, 24 rue Lhomond 75005 Paris, France
  Methods: biochemical methods
  
   
  
  Escherichia coli
  CSDB ID 20080 (all data & tools)

Show legend Show as text

Structure type: monomer
Contained glycoepitopes: IEDB_137353,IEDB_140947,IEDB_2189046

• Article ID: 3270
  De Leon GP, Elowe NH, Koteva KP, Valvano MA, Wright GD "An In Vitro Screen of Bacterial Lipopolysaccharide Biosynthetic Enzymes Identifies an Inhibitor of ADP-Heptose Biosynthesis" - Chemistry and Biology 13(4) (2006) 437-441
  

  The lipopolysaccharide (LPS)-rich outer membrane of gram-negative bacteria provides a protective barrier that insulates these organisms from the action of numerous antibiotics. Breach of the LPS layer can therefore provide access to the cell interior to otherwise impermeant toxic molecules and can expose vulnerable binding sites for immune system components such as complement. Inhibition of LPS biosynthesis, leading to a truncated LPS molecule, is an alternative strategy for antibacterial drug development in which this vital cellular structure is weakened. A significant challenge for in vitro screens of small molecules for inhibition of LPS biosynthesis is the difficulty in accessing the complex carbohydrate substrates. We have optimized an assay of the enzymes required for LPS heptose biosynthesis that simultaneously surveys five enzyme activities by using commercially available substrates and report its use in a small-molecule screen that identifies an inhibitor of heptose synthesis

  Lipopolysaccharide, biosynthesis, synthesis, LPS, structure, heptose, alternative, biosynthetic, Bacterial, carbohydrate, cell, molecule, Research, complex, bacteria, activity, biochemistry, Gram-negative bacteria, enzyme, gram negative bacteria, Gram-negative, cellular, inhibition, component, binding, binding site, site, Enzymes, action, membrane, substrate, heptose biosynthesis, protective, outer membrane, PDF, assay, immune, immune system, in vitro, use, challenge, development, drug, layer, complement, inhibitor, antibiotic, antimicrobial, antibacterial, toxic, Binding Sites, antibiotics, barrier

  NCBI PubMed ID: 16632256
  Journal NLM ID: 9500160
  Publisher: Maryland Heights, MO: Elsevier
  Correspondence: wrightge@mcmaster.ca
  Institutions: Antimicrobial Research Centre Department of Biochemistry and Biomedical Sciences McMaster University Hamilton, Hamilton, ON, Canada, Infectious Diseases Research Group Siebens-Drake Research Institute Department of Microbiology and Immunology The University of Western Ontario London, Ontario N6A 5C1 Canada
  Methods: biochemical methods
  
   
  
  Escherichia coli
  CSDB ID 20422 (all data & tools)

Show legend Show as text

Structure type: monomer
Trivial name: ADP-β-D-mannose
Contained glycoepitopes: IEDB_137353,IEDB_137485,IEDB_140947,IEDB_144983,IEDB_152206,IEDB_983930,SB_44,SB_72

• Article ID: 3366
  Morrison JP, Tanner ME "A two-base mechanism for Escherichia coli ADP-L-glycero-D-manno-heptose 6-epimerase" - Biochemistry 46(12) (2007) 3916-3924
  

  ADP-L-glycero-D-manno-heptose 6-epimerase (HldD or AGME, formerly RfaD) catalyzes the inversion of configuration at C-6" of the heptose moiety of ADP-D-glycero-D-manno-heptose and ADP-L-glycero-D-manno-heptose. The epimerase HldD operates in the biosynthetic pathway of L-glycero-D-manno-heptose, which is a conserved sugar in the core region of lipopolysaccharide (LPS) of Gram-negative bacteria. Previous studies support a mechanism in which HldD uses its tightly bound NADP+ cofactor to oxidize directly at C-6", generating a ketone intermediate. A reduction of the ketone from the opposite face then occurs, generating the epimeric product. How the epimerase is able access both faces of the ketone intermediate with correct alignment of the three required components, NADPH, the ketone carbonyl, and a catalytic acid/base residue, is addressed here. It is proposed that the epimerase active site contains two catalytic pockets, each of which bears a catalytic acid/base residue that facilitates reduction of the C-6" ketone but leads to a distinct epimeric product. The ketone carbonyl may access either pocket via rotation about the C-5"-C-6" bond of the sugar nucleotide and in doing so presents opposing faces to the bound cofactor. Evidence in support of the two-base mechanism is found in studies of two single mutants of the Escherichia coli K-12 epimerase, Y140F and K178M, both of which have severely compromised epimerase activities that are more than 3 orders of magnitude lower than that of the wild type. The catalytic competency of these two mutants in promoting redox chemistry is demonstrated with an alternate catalytic activity that requires only one catalytic base: dismutation of a C-6" aldehyde substrate analogue (ADP-β-D-manno-hexodialdose) to an acid and an alcohol (ADP-β-d-mannuronic acid and ADP-β-D-mannose). This study identifies the two catalytic bases as tyrosine 140 and lysine 178. A one-step enzymatic conversion of mannose into ADP-β-mannose is also described and used to make C-6"-substituted derivatives of this sugar nucleotide

  Lipopolysaccharide, biosynthesis, L-glycero-D-manno-heptose, epimerase, sugar nucleotide, Escherichia coli K12

  NCBI PubMed ID: 17316025
  Journal NLM ID: 0370623
  Publisher: American Chemical Society
  Correspondence: mtanner@chem.ubc.ca
  Institutions: Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1
  Methods: 1H NMR, 31P NMR, ESI-MS, chemical methods, genetic methods
  
   
  
  Escherichia coli K12 W3110
  CSDB ID 21868 (all data & tools)

Show legend Show as text

Structure type: monomer
Trivial name: ADP-D-glycero-D-manno-heptose (ADP-DDHep), ADP-D-glycero-β-D-manno-heptose
Contained glycoepitopes: IEDB_137353,IEDB_140947

• Article ID: 3371
  Mayer A, Tanner ME "Intermediate release by ADP-L-glycero-D-manno-heptose 6-epimerase" - Biochemistry 46(20) (2007) 6149-6155
  

  ADP-l-glycero-d-manno-heptose 6-epimerase (HldD or AGME, formerly RfaD) catalyzes the interconversion of ADP-β-D-glycero-D-manno-heptose (ADP-d,d-Hep) and ADP-β-L-glycero-d-manno-heptose (ADP-l,d-Hep). The latter compound provides the heptose moiety that is used in lipopolysaccharide biosynthesis by Gram-negative bacteria. Several lines of evidence suggest that the enzyme uses a direct oxidation/reduction mechanism involving a tightly bound NADP+ cofactor. An initial oxidation at C-6' ' gives a 6' '-keto intermediate, and a subsequent reduction on the opposite face of the carbonyl group generates the epimeric product. The reorientation required for the nonstereoselective reduction could take place within a single active site, or it could involve the release of the intermediate and rebinding in an altered conformation. To distinguish between these possibilities, two isotopically labeled substrates (ADP-d,d-Hep) were prepared that contained 18O and 2H isotopes at C-7' ' and C-6' ', respectively. A crossover experiment was used to determine whether unlabeled or doubly labeled products were formed upon epimerization of a mixture of the two singly labeled compounds. After an initial epimeric equilibrium was reached, no crossover could be detected, indicating that intermediate release is not intrinsic to the overall mechanism. After extended incubation, however, scrambling of the labels could be detected, indicating that a low background rate of intermediate release does occur. To directly detect the release of the intermediate, the labeled compounds were independently epimerized in the presence of a ketone-trapping reagent, phenylhydrazine. The corresponding phenylhydrazones were identified by mass spectrometry, and the absence of any 2H isotope in the adduct obtained from the deuterated starting compound confirmed that the oxidation had occurred at C-6'.

  Lipopolysaccharide, biosynthesis, heptose, gram negative bacteria, rfaD, epimerization

  NCBI PubMed ID: 17455913
  Journal NLM ID: 0370623
  Publisher: American Chemical Society
  Correspondence: mtanner@chem.ubc.ca
  Institutions: Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1
  Methods: 1H NMR, 31P NMR, ESI-MS, genetic methods, biochemical methods, isotope crossover
  
   
  
  Escherichia coli K12
  CSDB ID 21506 (all data & tools)
• Article ID: 3596
  Desroy N, Moreau F, Briet S, Fralliec GL, Floquet S, Durant L, Vongsouthi V, Gerusz V, Denis A, Escaich S "Towards Gram-negative antivirulence drugs: New inhibitors of HldE kinase" - Bioorganic and Medicinal Chemistry 17(3) (2008) 1276-1289
  

  Gram-negative bacteria lacking heptoses in their lipopolysaccharide (LPS) display attenuated virulence and increased sensitivity to human serum and to some antibiotics. Thus inhibition of bacterial heptose synthesis represents an attractive target for the development of new antibacterial agents. HldE is a bifunctional enzyme involved in the synthesis of bacterial heptoses. Development of a biochemical assay suitable for high-throughput screening allowed the discovery of inhibitors 1 and 2 of HldE kinase. Study of the structure-activity relationship of this series of inhibitors led to highly potent compounds.

  Lipopolysaccharide, Escherichia coli, antibacterial, antivirulence drugs, membrane permeability, HldE, RfaE

  NCBI PubMed ID: 19124251
  Journal NLM ID: 9413298
  Publisher: Elsevier
  Correspondence: N. Desroy
  Institutions: MUTABILIS SA, 102 Avenue Gaston Roussel, 93230 Romainville, France
  Methods: 1H NMR, ESI-MS, biochemical methods
  
   
  
  Escherichia coli
  CSDB ID 23512 (all data & tools)