N-Acetylmuramic Acid

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Christoph Mayer - One of the best experts on this subject based on the ideXlab platform.

  • Peptidoglycan-type analysis of the N-Acetylmuramic Acid auxotrophic oral pathogen Tannerella forsythia and reclassification of the peptidoglycan-type of Porphyromonas gingivalis
    BMC microbiology, 2019
    Co-Authors: Valentina M. T. Mayer, Isabel Hottmann, Christoph Mayer, Rudolf Figl, Friedrich Altmann, Christina Schäffer
    Abstract:

    Tannerella forsythia is a Gram-negative oral pathogen. Together with Porphyromonas gingivalis and Treponema denticola it constitutes the “red complex” of bacteria, which is crucially associated with periodontitis, an inflammatory disease of the tooth supporting tissues that poses a health burden worldwide. Due to the absence of common peptidoglycan biosynthesis genes, the unique bacterial cell wall sugar N-Acetylmuramic Acid (MurNAc) is an essential growth factor of T. forsythia to build up its peptidoglycan cell wall. Peptidoglycan is typically composed of a glycan backbone of alternating N-acetylglucosamine (GlcNAc) and MurNAc residues that terminates with anhydroMurNAc (anhMurNAc), and short peptides via which the sugar backbones are cross-linked to build up a bag-shaped network. We investigated T. forsythia’s peptidoglycan structure, which is an essential step towards anti-infective strategies against this pathogen. A new sensitive radioassay was developed which verified the presence of MurNAc and anhMurNAc in the cell wall of the bacterium. Upon digest of isolated peptidoglycan with endo-N-acetylmuramidase, exo-N-acetylglucosaminidase and muramyl-L-alanine amidase, respectively, peptidoglycan fragments were obtained. HPLC and mass spectrometry (MS) analyses revealed the presence of GlcNAc-MurNAc-peptides and the cross-linked dimer with retention-times and masses, respectively, equalling those of control digests of Escherichia coli and P. gingivalis peptidoglycan. Data were confirmed by tandem mass spectrometry (MS2) analysis, revealing the GlcNAc-MurNAc-tetra-tetra-MurNAc-GlcNAc dimer to contain the sequence of the amino Acids alanine, glutamic Acid, diaminopimelic Acid (DAP) and alanine, as well as a direct cross-link between DAP on the third and alanine on the fourth position of the two opposite stem peptides. The stereochemistry of DAP was determined by reversed-phase HPLC after dabsylation of hydrolysed peptidoglycan to be of the meso-type. T. forsythia peptidoglycan is of the A1γ-type like that of E. coli. Additionally, the classification of P. gingivalis peptidoglycan as A3γ needs to be revised to A1γ, due to the presence of meso-DAP instead of LL-DAP, as reported previously.

  • N-Acetylmuramic Acid (MurNAc) Auxotrophy of the Oral Pathogen Tannerella forsythia: Characterization of a MurNAc Kinase and Analysis of Its Role in Cell Wall Metabolism
    Frontiers in microbiology, 2018
    Co-Authors: Isabel Hottmann, Christina Schäffer, Matthew B. Calvert, Alexander Titz, Valentina M. T. Mayer, Markus B. Tomek, Valentin Friedrich, Christoph Mayer
    Abstract:

    Tannerella forsythia is an anaerobic, Gram-negative oral pathogen that thrives in multispecies gingival biofilms associated with periodontitis. The bacterium is auxotrophic for the commonly essential bacterial cell wall sugar N-Acetylmuramic Acid (MurNAc) and, thus, strictly depends on an exogenous supply of MurNAc for growth and maintenance of cell morphology. A MurNAc transporter (Tf_MurT; Tanf_08375) and an ortholog of the Escherichia coli etherase MurQ (Tf_MurQ; Tanf_08385) converting MurNAc-6-phosphate to GlcNAc-6-phosphate were recently described for T. forsythia. In between the respective genes on the T. forsythia genome, a putative kinase gene is located. In this study, the putative kinase (Tf_MurK; Tanf_08380) was produced as a recombinant protein and biochemically characterized. Kinetic studies revealed Tf_MurK to be a 6-kinase with stringent substrate specificity for MurNAc exhibiting a 6 × 104-fold higher catalytic efficiency (kcat/Km ) for MurNAc than for N-acetylglucosamine (GlcNAc) with kcat values of 10.5 s-1 and 0.1 s-1 and Km values of 200 μM and 116 mM, respectively. The enzyme kinetic data suggest that Tf_MurK is subject to substrate inhibition (Ki[S] = 4.2 mM). To assess the role of Tf_MurK in the cell wall metabolism of T. forsythia, a kinase deletion mutant (ΔTf_murK::erm) was constructed. This mutant accumulated MurNAc intracellularly in the exponential phase, indicating the capability to take up MurNAc, but inability to catabolize MurNAc. In the stationary phase, the MurNAc level was reduced in the mutant, while the level of the peptidoglycan precursor UDP-MurNAc-pentapeptide was highly elevated. Further, according to scanning electron microscopy evidence, the ΔTf_murK::erm mutant was more tolerant toward low MurNAc concentration in the medium (below 0.5 μg/ml) before transition from healthy, rod-shaped to fusiform cells occurred, while the parent strain required > 1 μg/ml MurNAc for optimal growth. These data reveal that T. forsythia readily catabolizes exogenous MurNAc but simultaneously channels a proportion of the sugar into peptidoglycan biosynthesis. Deletion of Tf_murK blocks MurNAc catabolism and allows the direction of MurNAc solely to peptidoglycan biosynthesis, resulting in a growth advantage in MurNAc-depleted medium. This work increases our understanding of the T. forsythia cell wall metabolism and may pave new routes for lead finding in the treatment of periodontitis.

  • An efficient synthesis of 1,6-anhydro-N-Acetylmuramic Acid from N-acetylglucosamine.
    Beilstein journal of organic chemistry, 2017
    Co-Authors: Matthew B. Calvert, Christoph Mayer, Alexander Titz
    Abstract:

    A novel synthesis of 1,6-anhydro-N-Acetylmuramic Acid is described, which proceeds in only five steps from the cheap starting material N-acetylglucosamine. This efficient synthesis should enable future studies into the importance of 1,6-anhydromuramic Acid in bacterial cell wall recycling processes.

  • Enzymatic synthesis and semi-preparative isolation of N-Acetylmuramic Acid 6-phosphate.
    Carbohydrate research, 2017
    Co-Authors: Sandra Unsleber, Marina Borisova, Christoph Mayer
    Abstract:

    Abstract N-Acetylmuramic Acid 6-phosphate (MurNAc-6P) is a constituent of the bacterial peptidoglycan cell wall, serving as an anchor point of secondary cell wall polymers such as teichoic Acids, and it is a key metabolite of the peptidoglycan recycling metabolism. Thus, there is a demand for MurNAc-6P as a standard for cell wall compositional and metabolic analyses and, in addition, as a substrate for peptidoglycan recycling enzymes, e.g. MurNAc-6P etherases (MurQ) and MurNAc-6P phosphatases (MupP), or as an effector molecule of transcriptional MurR regulators. However, MurNAc-6P is commercially not available. We report here the facile enzymatic production of MurNAc-6P in mg-scale from MurNAc and ATP, applying Clostridium acetobutylicum kinase MurK, and purification by semi-preparative HPLC. MurNAc-6P was quantified using a coupled enzyme assay, revealing 75–80% overall product yield, and high purity was confirmed by mass spectrometry and proton NMR.

  • The N-Acetylmuramic Acid 6-Phosphate Phosphatase MupP Completes the Pseudomonas Peptidoglycan Recycling Pathway Leading to Intrinsic Fosfomycin Resistance
    mBio, 2017
    Co-Authors: Marina Borisova, Jonathan Gisin, Christoph Mayer
    Abstract:

    Bacterial cells are encased in and stabilized by a netlike peptidoglycan (PGN) cell wall that undergoes turnover during bacterial growth. PGN turnover fragments are frequently salvaged by the cells via a pathway referred to as PGN recycling. Two different routes for the recycling of the cell wall sugar N-Acetylmuramic Acid (MurNAc) have been recognized in bacteria. In Escherichia coli and related enterobacteria, as well as in most Gram-positive bacteria, MurNAc is recovered via a catabolic route requiring a MurNAc 6-phosphate etherase (MurQ in E. coli) enzyme. However, many Gram-negative bacteria, including Pseudomonas species, lack a MurQ ortholog and use an alternative, anabolic recycling route that bypasses the de novo biosynthesis of uridyldiphosphate (UDP)-MurNAc, the first committed precursor of PGN. Bacteria featuring the latter pathway become intrinsically resistant to the antibiotic fosfomycin, which targets the de novo biosynthesis of UDP-MurNAc. We report here the identification and characterization of a phosphatase enzyme, named MupP, that had been predicted to complete the anabolic recycling pathway of Pseudomonas species but has remained unknown so far. It belongs to the large haloAcid dehalogenase family of phosphatases and specifically converts MurNAc 6-phosphate to MurNAc. A ΔmupP mutant of Pseudomonas putida was highly susceptible to fosfomycin, accumulated large amounts of MurNAc 6-phosphate, and showed lower levels of UDP-MurNAc than wild-type cells, altogether consistent with a role for MupP in the anabolic PGN recycling route and as a determinant of intrinsic resistance to fosfomycin.IMPORTANCE Many Gram-negative bacteria, but not E. coli, make use of a cell wall salvage pathway that contributes to the pool of UDP-MurNAc, the first committed precursor of cell wall synthesis in bacteria. This salvage pathway is of particular interest because it confers intrinsic resistance to the antibiotic fosfomycin, which blocks de novo UDP-MurNAc biosynthesis. Here we identified and characterized a previously missing enzyme within the salvage pathway, the MurNAc 6-phosphate phosphatase MupP of P. putida MupP, together with the other enzymes of the anabolic recycling pathway, AnmK, AmgK, and MurU, yields UDP-MurNAc, renders bacteria intrinsically resistant to fosfomycin, and thus may serve as a novel drug target for antimicrobial therapy.

Didier Blanot - One of the best experts on this subject based on the ideXlab platform.

  • The peptidoglycan sacculus of Myxococcus xanthus has unusual structural features and is degraded during glycerol-induced myxospore development.
    Journal of bacteriology, 2008
    Co-Authors: Nhat Khai Bui, Didier Blanot, Joe Gray, Heinz Schwarz, Peter Schumann, Waldemar Vollmer
    Abstract:

    Upon nutrient limitation cells of the swarming soil bacterium Myxococcus xanthus form a multicellular fruiting body in which a fraction of the cells develop into myxospores. Spore development includes the transition from a rod-shaped vegetative cell to a spherical myxospore and so is expected to be accompanied by changes in the bacterial cell envelope. Peptidoglycan is the shape-determining structure in the cell envelope of most bacteria, including myxobacteria. We analyzed the composition of peptidoglycan isolated from M. xanthus. While the basic structural elements of peptidoglycan in myxobacteria were identical to those in other gram-negative bacteria, the peptidoglycan of M. xanthus had unique structural features. meso- or LL-diaminopimelic Acid was present in the stem peptides, and a new modification of N-Acetylmuramic Acid was detected in a fraction of the muropeptides. Peptidoglycan formed a continuous, bag-shaped sacculus in vegetative cells. The sacculus was degraded during the transition from vegetative cells to glycerol-induced myxospores. The spherical, bag-shaped coats isolated from glycerol-induced spores contained no detectable muropeptides, but they contained small amounts of N-Acetylmuramic Acid and meso-diaminopimelic Acid.

  • cytoplasmic steps of peptidoglycan biosynthesis
    Fems Microbiology Reviews, 2008
    Co-Authors: Helene Barreteau, Andreja Kovac, Audrey Boniface, Matej Sova, Stanislav Gobec, Didier Blanot
    Abstract:

    The biosynthesis of bacterial cell wall peptidoglycan is a complex process that involves enzyme reactions that take place in the cytoplasm (synthesis of the nucleotide precursors) and on the inner side (synthesis of lipid-linked intermediates) and outer side (polymerization reactions) of the cytoplasmic membrane. This review deals with the cytoplasmic steps of peptidoglycan biosynthesis, which can be divided into four sets of reactions that lead to the syntheses of (1) UDP-N-acetylglucosamine from fructose 6-phosphate, (2) UDP-N-Acetylmuramic Acid from UDP-N-acetylglucosamine, (3) UDP-N-acetylmuramyl-pentapeptide from UDP-N-Acetylmuramic Acid and (4) D-glutamic Acid and dipeptide D-alanyl-D-alanine. Recent data concerning the different enzymes involved are presented. Moreover, special attention is given to (1) the chemical and enzymatic synthesis of the nucleotide precursor substrates that are not commercially available and (2) the search for specific inhibitors that could act as antibacterial compounds.

  • Study of the overproduced uridine-diphosphate-N-acetylmuramate:L-alanine ligase from Escherichia coli.
    Microbial drug resistance (Larchmont N.Y.), 1996
    Co-Authors: Dominique Liger, Jean Van Heijenoort, Didier Blanot, Anne Masson, Claudine Parquet
    Abstract:

    The UDP-N-acetylmuramate:L-alanine ligase of Escherichia coli is responsible for the addition of the first amino Acid of the peptide moiety in the assembly of the monomer unit of peptidoglycan. It catalyzes the formation of the amide bond between UDP-N-Acetylmuramic Acid (UDP-MurNAc) and L-alanine. The UDP-MurNAc-L-alanine ligase was overproduced 2000-fold in a strain harboring a recombinant plasmid (pAM1005) with the murC gene under the control of the inducible promoter trc. The murC gene product appears as a 50-kDa protein accounting for ca. 50% of total cell proteins. A two-step purification led to 1 g of a homogeneous protein from an 8-liter culture. The N-terminal sequence of the purified protein correlated with the nucleotide sequence of the gene. The stability of the enzymatic activity is strictly dependent on the presence of 2-mercaptoethanol. The K(m) values for substrates UDP-N-Acetylmuramic Acid, L-alanine, and ATP were estimated; 100, 20, and 450 microM, respectively. The specificity of the enzyme for its substrates was investigated with various analogues. Preliminary experiments attempting to elucidate the enzymatic mechanism were consistent with the formation of an acylphosphate intermediate.

  • Synthesis of α and β Anomers of UDP-N-Acetylmuramic Acid
    Carbohydrate Research, 1994
    Co-Authors: Didier Blanot, Geneviève Auger, Dominique Liger, Jean Van Heijenoort
    Abstract:

    UDP-N-acetylmuramyl (UDP-MurNAc) derivatives are substrates for several cytoplasmics steps of the synthesis of bacterial peptidoglycan (Park, 1952). Their availability is a prerequisite for developing the detailed study of the synthetases catalyzing these reactions. Since they are not commercial compounds, they have to be prepared from bacterial cells in which they accumulate under specific conditions. However, such procedures are long and tedious, and yields are often low. An alternative approach is to chemically synthesize UDP-N-Acetylmuramic Acid on a large scale, and to use it as starting material for the in vitro enzymatic preparation of the other UDP-MurNAc precursors. In this communication, we wish to report the total synthesis of UDP-MurNAc.

Christopher T Walsh - One of the best experts on this subject based on the ideXlab platform.

  • (E)-enolbutyryl-UDP-N-acetylglucosamine as a mechanistic probe of UDP-N-acetylenolpyruvylglucosamine reductase (MurB).
    Biochemistry, 1996
    Co-Authors: Watson J. Lees, James M Hogle, Timothy E Benson, Christopher T Walsh
    Abstract:

    UDP-N-acetylenolpyruvylglucosamine reductase (MurB), a peptidoglycan biosynthetic enzyme from Escherichia coli, reduces both (E)- and (Z)-isomers of enolbutyryl-UDP-GlcNAc, C4 analogs of the physiological C3 enolpyruvyl substrate, to UDP-methyl-N-Acetylmuramic Acid in the presence of NADPH. The X-ray crystal structure of the (E)-enolbutyryl-UDP-GlcNAc-MurB complex is similar to that of the enolpyruvyl-UDP-GlcNAc-MurB complex. In both structures the groups thought to be involved in hydride transfer to C3 and protonation at C2 of the enol ether substrate are arranged anti relative to the enol double bond. The stereochemical outcome of reduction of (E)-enolbutyryl-UDP-GlcNAc by NADPD in D2O is thus predicted to yield a (2R,3R)-dideuterio product. This was validated by conversion of the 2,3-dideuterio-UDP-methyl-N-Acetylmuramic Acid product to 2,3-dideuterio-2-hydroxybutyrate, which was shown to be (2R) by enzymatic analysis and (3R) by NMR comparison to authentic (2R,3R)- and (2R,3S)-2,3-dideuterio-2-hydroxybutyrate. Remarkably, the (E)-enolbutyryl-UDP-GlcNAc was found to partition between reduction to UDP-methyl-N-Acetylmuramic and isomerization to the (Z)-substrate isomer in the MurB active site, indicative of a C2 carbanion/enol species that is sufficiently long-lived to rotate around the C2-C3 single bond during catalysis.

  • An enzyme–substrate complex involved in bacterial cell wall biosynthesis
    Nature Structural Biology, 1995
    Co-Authors: Timothy E Benson, David J Filman, Christopher T Walsh, James M Hogle
    Abstract:

    The crystal structure of UDP- N -acetylenolpyruvylglucosamine reductase in the presence of its substrate, enolpyruvyl-UDP- N -acetylglucosamine, has been solved to 2.7 Å resolution. This enzyme is responsible for the synthesis of UDP- N -acetylmuramic Acid in bacterial cell wall biosynthesis and consequently provides an attractive target for the design of antibacterial agents. The structure reveals a novel flavin binding motif, shows a striking alignment of the flavin with the substrate, and suggests a catalytic mechanism for the reduction of this unusual enol ether.

  • an enzyme substrate complex involved in bacterial cell wall biosynthesis
    Nature Structural & Molecular Biology, 1995
    Co-Authors: Timothy E Benson, David J Filman, Christopher T Walsh, James M Hogle
    Abstract:

    The crystal structure of UDP-N-acetylenolpyruvylglucosamine reductase in the presence of its substrate, enolpyruvyl-UDP-N-acetylglucosamine, has been solved to 2.7 A resolution. This enzyme is responsible for the synthesis of UDP-N-Acetylmuramic Acid in bacterial cell wall biosynthesis and consequently provides an attractive target for the design of antibacterial agents. The structure reveals a novel flavin binding motif, shows a striking alignment of the flavin with the substrate, and suggests a catalytic mechanism for the reduction of this unusual enol ether.

  • Overexpression, purification, and mechanistic study of UDP-N-acetylenolpyruvylglucosamine reductase
    Biochemistry, 1993
    Co-Authors: Timothy E Benson, John L. Marquardt, Anne C. Marquardt, Felicia A. Etzkorn, Christopher T Walsh
    Abstract:

    The recently isolated Escherichia coli murB gene (Pucci et al., 1992) has been cloned into an expression vector and the encoded UDP-N-acetylenolpyruvylglucosamine reductase (EC 1.1.1.158) was overproduced to about 10% of soluble cell protein. The encoded 38-kDa protein has been purified to near homogeneity. It was found to be a monomer and to contain stoichiometric amounts of bound FAD which is reducible in catalytic turnover. The enzyme utilizes the 4-pro-S hydrogen of NADPH to reduce the enolpyruvyl group of UDP-N-acetylglucosamine enolpyruvate to the lactyl ether in UDP-N-Acetylmuramic Acid. NMR analysis of products from 2H2O and 4S-[2H]NADPH incubations establishes that a hydride from NADPH via E.FADH2 is transferred to the beta-methyl of the 3-O-lactyl moiety and a proton from solvent to the alpha-carbon of the lactyl moiety of UDP-N-Acetylmuramic Acid. A mechanism for this unusual enolether reduction in bacterial cell wall assembly is proposed.

Christina Schäffer - One of the best experts on this subject based on the ideXlab platform.

  • Peptidoglycan-type analysis of the N-Acetylmuramic Acid auxotrophic oral pathogen Tannerella forsythia and reclassification of the peptidoglycan-type of Porphyromonas gingivalis
    BMC microbiology, 2019
    Co-Authors: Valentina M. T. Mayer, Isabel Hottmann, Christoph Mayer, Rudolf Figl, Friedrich Altmann, Christina Schäffer
    Abstract:

    Tannerella forsythia is a Gram-negative oral pathogen. Together with Porphyromonas gingivalis and Treponema denticola it constitutes the “red complex” of bacteria, which is crucially associated with periodontitis, an inflammatory disease of the tooth supporting tissues that poses a health burden worldwide. Due to the absence of common peptidoglycan biosynthesis genes, the unique bacterial cell wall sugar N-Acetylmuramic Acid (MurNAc) is an essential growth factor of T. forsythia to build up its peptidoglycan cell wall. Peptidoglycan is typically composed of a glycan backbone of alternating N-acetylglucosamine (GlcNAc) and MurNAc residues that terminates with anhydroMurNAc (anhMurNAc), and short peptides via which the sugar backbones are cross-linked to build up a bag-shaped network. We investigated T. forsythia’s peptidoglycan structure, which is an essential step towards anti-infective strategies against this pathogen. A new sensitive radioassay was developed which verified the presence of MurNAc and anhMurNAc in the cell wall of the bacterium. Upon digest of isolated peptidoglycan with endo-N-acetylmuramidase, exo-N-acetylglucosaminidase and muramyl-L-alanine amidase, respectively, peptidoglycan fragments were obtained. HPLC and mass spectrometry (MS) analyses revealed the presence of GlcNAc-MurNAc-peptides and the cross-linked dimer with retention-times and masses, respectively, equalling those of control digests of Escherichia coli and P. gingivalis peptidoglycan. Data were confirmed by tandem mass spectrometry (MS2) analysis, revealing the GlcNAc-MurNAc-tetra-tetra-MurNAc-GlcNAc dimer to contain the sequence of the amino Acids alanine, glutamic Acid, diaminopimelic Acid (DAP) and alanine, as well as a direct cross-link between DAP on the third and alanine on the fourth position of the two opposite stem peptides. The stereochemistry of DAP was determined by reversed-phase HPLC after dabsylation of hydrolysed peptidoglycan to be of the meso-type. T. forsythia peptidoglycan is of the A1γ-type like that of E. coli. Additionally, the classification of P. gingivalis peptidoglycan as A3γ needs to be revised to A1γ, due to the presence of meso-DAP instead of LL-DAP, as reported previously.

  • N-Acetylmuramic Acid (MurNAc) Auxotrophy of the Oral Pathogen Tannerella forsythia: Characterization of a MurNAc Kinase and Analysis of Its Role in Cell Wall Metabolism
    Frontiers in microbiology, 2018
    Co-Authors: Isabel Hottmann, Christina Schäffer, Matthew B. Calvert, Alexander Titz, Valentina M. T. Mayer, Markus B. Tomek, Valentin Friedrich, Christoph Mayer
    Abstract:

    Tannerella forsythia is an anaerobic, Gram-negative oral pathogen that thrives in multispecies gingival biofilms associated with periodontitis. The bacterium is auxotrophic for the commonly essential bacterial cell wall sugar N-Acetylmuramic Acid (MurNAc) and, thus, strictly depends on an exogenous supply of MurNAc for growth and maintenance of cell morphology. A MurNAc transporter (Tf_MurT; Tanf_08375) and an ortholog of the Escherichia coli etherase MurQ (Tf_MurQ; Tanf_08385) converting MurNAc-6-phosphate to GlcNAc-6-phosphate were recently described for T. forsythia. In between the respective genes on the T. forsythia genome, a putative kinase gene is located. In this study, the putative kinase (Tf_MurK; Tanf_08380) was produced as a recombinant protein and biochemically characterized. Kinetic studies revealed Tf_MurK to be a 6-kinase with stringent substrate specificity for MurNAc exhibiting a 6 × 104-fold higher catalytic efficiency (kcat/Km ) for MurNAc than for N-acetylglucosamine (GlcNAc) with kcat values of 10.5 s-1 and 0.1 s-1 and Km values of 200 μM and 116 mM, respectively. The enzyme kinetic data suggest that Tf_MurK is subject to substrate inhibition (Ki[S] = 4.2 mM). To assess the role of Tf_MurK in the cell wall metabolism of T. forsythia, a kinase deletion mutant (ΔTf_murK::erm) was constructed. This mutant accumulated MurNAc intracellularly in the exponential phase, indicating the capability to take up MurNAc, but inability to catabolize MurNAc. In the stationary phase, the MurNAc level was reduced in the mutant, while the level of the peptidoglycan precursor UDP-MurNAc-pentapeptide was highly elevated. Further, according to scanning electron microscopy evidence, the ΔTf_murK::erm mutant was more tolerant toward low MurNAc concentration in the medium (below 0.5 μg/ml) before transition from healthy, rod-shaped to fusiform cells occurred, while the parent strain required > 1 μg/ml MurNAc for optimal growth. These data reveal that T. forsythia readily catabolizes exogenous MurNAc but simultaneously channels a proportion of the sugar into peptidoglycan biosynthesis. Deletion of Tf_murK blocks MurNAc catabolism and allows the direction of MurNAc solely to peptidoglycan biosynthesis, resulting in a growth advantage in MurNAc-depleted medium. This work increases our understanding of the T. forsythia cell wall metabolism and may pave new routes for lead finding in the treatment of periodontitis.

  • identification of a novel n acetylmuramic Acid transporter in tannerella forsythia
    Journal of Bacteriology, 2016
    Co-Authors: Angela Ruscitto, Isabel Hottmann, Christina Schäffer, Graham P Stafford, Christoph Mayer, Ashu Sharma
    Abstract:

    ABSTRACT Tannerella forsythia is a Gram-negative periodontal pathogen lacking the ability to undergo de novo synthesis of amino sugars N -acetylmuramic Acid (MurNAc) and N -acetylglucosamine (GlcNAc) that form the disaccharide repeating unit of the peptidoglycan backbone. T. forsythia relies on the uptake of these sugars from the environment, which is so far unexplored. Here, we identified a novel transporter system of T. forsythia involved in the uptake of MurNAc across the inner membrane and characterized a homolog of the Escherichia coli MurQ etherase involved in the conversion of MurNAc-6-phosphate (MurNAc-6-P) to GlcNAc-6-P. The genes encoding these components were identified on a three-gene cluster spanning Tanf_08375 to Tanf_08385 located downstream from a putative peptidoglycan recycling locus. We show that the three genes, Tanf_08375, Tanf_08380, and Tanf_08385, encoding a MurNAc transporter, a putative sugar kinase, and a MurQ etherase, respectively, are transcriptionally linked. Complementation of the Tanf_08375 and Tanf_08380 genes together in trans , but not individually, rescued the inability of an E. coli mutant deficient in the phosphotransferase (PTS) system-dependent MurNAc transporter MurP as well as that of a double mutant deficient in MurP and components of the PTS system to grow on MurNAc. In addition, complementation with this two-gene construct in E. coli caused depletion of MurNAc in the medium, further confirming this observation. Our results show that the products of Tanf_08375 and Tanf_08380 constitute a novel non-PTS MurNAc transporter system that seems to be widespread among bacteria of the Bacteroidetes phylum. To the best of our knowledge, this is the first identification of a PTS-independent MurNAc transporter in bacteria. IMPORTANCE In this study, we report the identification of a novel transporter for peptidoglycan amino sugar N -acetylmuramic Acid (MurNAc) in the periodontal pathogen T. forsythia. It has been known since the late 1980s that T. forsythia is a MurNAc auxotroph relying on environmental sources for this essential sugar. Most sugar transporters, and the MurNAc transporter MurP in particular, require a PTS phosphorelay to drive the uptake and concurrent phosphorylation of the sugar through the inner membrane in Gram-negative bacteria. Our study uncovered a novel type of PTS-independent MurNAc transporter, and although so far, it seems to be unique to T. forsythia, it may be present in a range of bacteria both of the oral cavity and gut, especially of the phylum Bacteroidetes.

  • N-Acetylmuramic Acid as Capping Element of α-D-Fucose-containing S-layer Glycoprotein Glycans from Geobacillus tepidamans GS5–97T
    The Journal of biological chemistry, 2005
    Co-Authors: Hanspeter Kählig, Christina Schäffer, Daniel Kolarich, Sonja Zayni, Andrea Scheberl, Paul Kosma, Paul Messner
    Abstract:

    Abstract Geobacillus tepidamans GS5–97T is a novel Gram-positive, moderately thermophilic bacterial species that is covered by a glycosylated surface layer (S-layer) protein. The isolated and purified S-layer glycoprotein SgtA was ultrastructurally and chemically investigated and showed several novel properties. By SDS-PAGE, SgtA was separated into four distinct bands in an apparent molecular mass range of 106–166 kDa. The three high molecular mass bands gave a positive periodic Acid-Schiff staining reaction, whereas the 106-kDa band was nonglycosylated. Glycosylation of SgtA was investigated by means of chemical analyses, 600-MHz nuclear magnetic resonance spectroscopy, and electrospray ionization quadrupole time-of-fight mass spectrometry. Glycopeptides obtained after Pronase digestion revealed the glycan structure [→2)-α-l-Rhap-(1→3)-α-d-Fucp-(1→]n = ∼ 20, with d-fucopyranose having never been identified before as a constituent of S-layer glycans. The rhamnose residue at the nonreducing end of the terminal repeating unit of the glycan chain was di-substituted. For the first time, (R)-N-Acetylmuramic Acid, the key component of prokaryotic peptidoglycan, was found in an α-linkage to carbon 3 of the terminal rhamnose residue, serving as capping motif of an S-layer glycan. In addition, that rhamnose was substituted at position 2 with a β-N-acetylglucosamine residue. The S-layer glycan chains were bound via the trisaccharide core →2)-α-l-Rhap-(1→3)-α-l-Rhap-(1→3)-α-l-Rhap-(1→ to carbon 3 of β-d-galactose, which was attached in O-glycosidic linkage to serine and threonine residues of SgtA of G. tepidamans GS5–97T.

Timothy E Benson - One of the best experts on this subject based on the ideXlab platform.

  • (E)-enolbutyryl-UDP-N-acetylglucosamine as a mechanistic probe of UDP-N-acetylenolpyruvylglucosamine reductase (MurB).
    Biochemistry, 1996
    Co-Authors: Watson J. Lees, James M Hogle, Timothy E Benson, Christopher T Walsh
    Abstract:

    UDP-N-acetylenolpyruvylglucosamine reductase (MurB), a peptidoglycan biosynthetic enzyme from Escherichia coli, reduces both (E)- and (Z)-isomers of enolbutyryl-UDP-GlcNAc, C4 analogs of the physiological C3 enolpyruvyl substrate, to UDP-methyl-N-Acetylmuramic Acid in the presence of NADPH. The X-ray crystal structure of the (E)-enolbutyryl-UDP-GlcNAc-MurB complex is similar to that of the enolpyruvyl-UDP-GlcNAc-MurB complex. In both structures the groups thought to be involved in hydride transfer to C3 and protonation at C2 of the enol ether substrate are arranged anti relative to the enol double bond. The stereochemical outcome of reduction of (E)-enolbutyryl-UDP-GlcNAc by NADPD in D2O is thus predicted to yield a (2R,3R)-dideuterio product. This was validated by conversion of the 2,3-dideuterio-UDP-methyl-N-Acetylmuramic Acid product to 2,3-dideuterio-2-hydroxybutyrate, which was shown to be (2R) by enzymatic analysis and (3R) by NMR comparison to authentic (2R,3R)- and (2R,3S)-2,3-dideuterio-2-hydroxybutyrate. Remarkably, the (E)-enolbutyryl-UDP-GlcNAc was found to partition between reduction to UDP-methyl-N-Acetylmuramic and isomerization to the (Z)-substrate isomer in the MurB active site, indicative of a C2 carbanion/enol species that is sufficiently long-lived to rotate around the C2-C3 single bond during catalysis.

  • An enzyme–substrate complex involved in bacterial cell wall biosynthesis
    Nature Structural Biology, 1995
    Co-Authors: Timothy E Benson, David J Filman, Christopher T Walsh, James M Hogle
    Abstract:

    The crystal structure of UDP- N -acetylenolpyruvylglucosamine reductase in the presence of its substrate, enolpyruvyl-UDP- N -acetylglucosamine, has been solved to 2.7 Å resolution. This enzyme is responsible for the synthesis of UDP- N -acetylmuramic Acid in bacterial cell wall biosynthesis and consequently provides an attractive target for the design of antibacterial agents. The structure reveals a novel flavin binding motif, shows a striking alignment of the flavin with the substrate, and suggests a catalytic mechanism for the reduction of this unusual enol ether.

  • an enzyme substrate complex involved in bacterial cell wall biosynthesis
    Nature Structural & Molecular Biology, 1995
    Co-Authors: Timothy E Benson, David J Filman, Christopher T Walsh, James M Hogle
    Abstract:

    The crystal structure of UDP-N-acetylenolpyruvylglucosamine reductase in the presence of its substrate, enolpyruvyl-UDP-N-acetylglucosamine, has been solved to 2.7 A resolution. This enzyme is responsible for the synthesis of UDP-N-Acetylmuramic Acid in bacterial cell wall biosynthesis and consequently provides an attractive target for the design of antibacterial agents. The structure reveals a novel flavin binding motif, shows a striking alignment of the flavin with the substrate, and suggests a catalytic mechanism for the reduction of this unusual enol ether.

  • Overexpression, purification, and mechanistic study of UDP-N-acetylenolpyruvylglucosamine reductase
    Biochemistry, 1993
    Co-Authors: Timothy E Benson, John L. Marquardt, Anne C. Marquardt, Felicia A. Etzkorn, Christopher T Walsh
    Abstract:

    The recently isolated Escherichia coli murB gene (Pucci et al., 1992) has been cloned into an expression vector and the encoded UDP-N-acetylenolpyruvylglucosamine reductase (EC 1.1.1.158) was overproduced to about 10% of soluble cell protein. The encoded 38-kDa protein has been purified to near homogeneity. It was found to be a monomer and to contain stoichiometric amounts of bound FAD which is reducible in catalytic turnover. The enzyme utilizes the 4-pro-S hydrogen of NADPH to reduce the enolpyruvyl group of UDP-N-acetylglucosamine enolpyruvate to the lactyl ether in UDP-N-Acetylmuramic Acid. NMR analysis of products from 2H2O and 4S-[2H]NADPH incubations establishes that a hydride from NADPH via E.FADH2 is transferred to the beta-methyl of the 3-O-lactyl moiety and a proton from solvent to the alpha-carbon of the lactyl moiety of UDP-N-Acetylmuramic Acid. A mechanism for this unusual enolether reduction in bacterial cell wall assembly is proposed.