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Susan M. Logan - One of the best experts on this subject based on the ideXlab platform.

  • Structural heterogeneity of carbohydrate modifications affects serospecificity of Campylobacter flagellins
    Molecular Microbiology, 2020
    Co-Authors: Susan M. Logan, Cheryl P Ewing, John F. Kelly, Pierre Thibault, Patricia Guerry
    Abstract:

    Summary Flagellin from Campylobacter coli VC167 is post-translationally modified at ≥ 16 amino Acid residues with Pseudaminic Acid and three related derivatives. The predominant modification was 5,7-diacetamido-3,5,7,9 - tetradeoxy - l - glycero - l - manno - nonulosonic Acid (Pseudaminic Acid, Pse5Ac7Ac), a modification that has been described previously on flagellin from Campylobacter jejuni 81-176. VC167 lacked two modi-fications present in 81-176 and instead had two unique modifications of masses 431 and 432 Da. Flagellins from both C. jejuni 81-176 and C. coli VC167 were also modified with an acetamidino form of Pseudaminic Acid (PseAm), but tandem mass spectrometry indicated that the structure of PseAm differed in the two strains. Synthesis of PseAm in C. coli VC167 requires a minimum of six ptm genes. In contrast, PseAm is synthesized in C. jejuni 81-176 via an alternative pathway using the product of the pseA gene. Mutation of the ptm genes in C. coli VC167 can be detected by changes in apparent Mr of flagellin in SDS-PAGE gels, changes in isoelectric focusing (IEF) patterns and loss of immunoreactivity with antiserum LAH2. These changes corresponded to loss of both 315 Da and 431 Da modifications from flagellin. Complementation of the VC167 ptm mutants with the 81-176 pseA gene in trans resulted in flagellins containing both 315 and 431 Da modifications, but these flagellins remained unreactive in LAH2 antibody, suggesting that the unique form of PseAm encoded by the ptm genes contributes to the serospecificity of the flagellar filament.

  • A novel glycan modifies the flagellar filament proteins of the oral bacterium Treponema denticola.
    Molecular Microbiology, 2016
    Co-Authors: Kurni Kurniyati, Susan M. Logan, John F. Kelly, Evgeny Vinogradov, Anna Robotham, Youbing Tu, Juyu Wang, Chunhao Li
    Abstract:

    While protein glycosylation has been reported in several spirochetes including the syphilis bacterium Treponema pallidum and Lyme disease pathogen Borrelia burgdorferi, the pertinent glycan structures and their roles remain uncharacterized. Herein, we report a novel glycan with an unusual chemical composition and structure in the oral spirochete Treponema denticola, a keystone pathogen of periodontitis. The identified glycan of mass 450.2 Da is composed of a monoacetylated nonulosonic Acid (Non) with a novel extended N7 acyl modification, a 2-methoxy-4,5,6-trihydroxy-hexanoyl residue in which the Non has a Pseudaminic Acid configuration (L-glycero-L-manno) and is β-linked to serine or threonine residues. This novel glycan modifies the flagellin proteins (FlaBs) of T. denticola by O-linkage at multiple sites near the D1 domain, a highly conserved region of bacterial flagellins that interact with Toll-like receptor 5. Furthermore, mutagenesis studies demonstrate that the glycosylation plays an essential role in the flagellar assembly and motility of T. denticola. To our knowledge, this novel glycan and its unique modification sites have not been reported previously in any bacteria. This article is protected by copyright. All rights reserved.

  • structural and functional analysis of campylobacter jejuni pseg a udp sugar hydrolase from the Pseudaminic Acid biosynthetic pathway
    Journal of Biological Chemistry, 2009
    Co-Authors: Erumbi S Rangarajan, Susan M. Logan, Dennis M. Whitfield, A Proteau, Zhanna Potetinova, Enrico O Purisima, Miroslaw Cygler, A Matte, Traian Sulea, Ian C. Schoenhofen
    Abstract:

    Flagella of the bacteria Helicobacter pylori and Campylobacter jejuni are important virulence determinants, whose proper assembly and function are dependent upon glycosylation at multiple positions by sialic Acid-like sugars, such as 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-nonulosonic Acid (Pseudaminic Acid (Pse)). The fourth enzymatic step in the Pseudaminic Acid pathway, the hydrolysis of UDP-2,4-diacetamido-2,4,6-trideoxy-β-l-altropyranose to generate 2,4-diacetamido-2,4,6-trideoxy-l-altropyranose, is performed by the nucleotide sugar hydrolase PseG. To better understand the molecular basis of the PseG catalytic reaction, we have determined the crystal structures of C. jejuni PseG in apo-form and as a complex with its UDP product at 1.8 and 1.85 Å resolution, respectively. In addition, molecular modeling was utilized to provide insight into the structure of the PseG-substrate complex. This modeling identifies a His17-coordinated water molecule as the putative nucleophile and suggests the UDP-sugar substrate adopts a twist-boat conformation upon binding to PseG, enhancing the exposure of the anomeric bond cleaved and favoring inversion at C-1. Furthermore, based on these structures a series of amino Acid substitution derivatives were constructed, altering residues within the active site, and each was kinetically characterized to examine its contribution to PseG catalysis. In conjunction with structural comparisons, the almost complete inactivation of the PseG H17F and H17L derivatives suggests that His17 functions as an active site base, thereby activating the nucleophilic water molecule for attack of the anomeric C–O bond of the UDP-sugar. As the PseG structure reveals similarity to those of glycosyltransferase family-28 members, in particular that of Escherichia coli MurG, these findings may also be of relevance for the mechanistic understanding of this important enzyme family.

  • The Engineering of Bacteria Bearing Azido-Pseudaminic Acid-Modified Flagella
    ChemBioChem, 2009
    Co-Authors: Annie Aubry, Ian C. Schoenhofen, Susan M. Logan, Martin E Tanner
    Abstract:

    Catch a tiger by the tail: We have demonstrated that by feeding nonmotile mutant C. jejuni bacteria with a neutral azide-labelled Pseudaminic Acid precursor we can restore their ability to generate functional flagella. The presence of azido-Pseudaminic Acid on the surface of the flagella provides a bio-orthogonal chemical handle that can be used to modify the flagellar proteins.

  • Identification of novel carbohydrate modifications on Campylobacter jejuni 11168 flagellin using metabolomics-based approaches.
    FEBS Journal, 2009
    Co-Authors: Susan M. Logan, Annie Aubry, John Kelly, Evgeny Vinogradov, Jeremy E. Melanson, Harald Nothaft
    Abstract:

    It is well known that the flagellin of Campylobacter jejuni is extensively glycosylated by Pseudaminic Acid and the related acetamindino derivative, in addition to flagellin glycosylation being essential for motility and colonization of host cells. Recently, the use of metabolomics permitted the unequivocal characterization of unique flagellin modifications in Campylobacter, including novel legionaminic Acid sugars in Campylobacter coli, which had been impossible to ascertain in earlier studies using proteomics-based approaches. To date, the precise identities of the flagellin glycosylation modifications have only been elucidated for C. jejuni 81-176 and C. coli VC167 and those present in the first genome-sequenced strain C. jejuni 11168 remain elusive due to lability and respective levels of individual glycan modifications. We report the characterization of the carbohydrate modifications on C. jejuni 11168 flagellin using metabolomics-based approaches. Detected as their corresponding CMP-linked precursors, structural information on the flagellin modifications was obtained using a combination of MS and NMR spectroscopy. In addition to the Pseudaminic Acid and legionaminic Acid sugars known to be present on Campylobacter flagellin, two unusual 2,3-di-O-methylglyceric Acid modifications of a nonulosonate sugar were identified. By performing a metabolomic analysis of selected isogenic mutants of genes from the flagellin glycosylation locus of this pathogen, these novel CMP-linked precursors were confirmed to be di-O-methylglyceric Acid derivatives of Pseudaminic Acid and the related acetamidino sugar. This is the first comprehensive analysis of the flagellar modifications in C. jejuni 11168 and structural elucidation of di-O-methylglyceric Acid derivatives of Pseudaminic Acid on Campylobacter flagellin.

Ian C. Schoenhofen - One of the best experts on this subject based on the ideXlab platform.

  • Utilizing CMP-Sialic Acid Analogs to Unravel Neisseria gonorrhoeae Lipooligosaccharide-Mediated Complement Resistance and Design Novel Therapeutics
    PLOS Pathogens, 2015
    Co-Authors: Sunita Gulati, Ian C. Schoenhofen, Dennis M. Whitfield, Evgeny Vinogradov, Jianjun Li, Frank St. Michael, Jacek Stupak, Bo Zheng, Makoto Ohnishi
    Abstract:

    Neisseria gonorrhoeae deploys a novel immune evasion strategy wherein the lacto-N-neotetraose (LNnT) structure of lipooligosaccharide (LOS) is capped by the bacterial sialyltransferase, using host cytidine-5’-monophosphate (CMP)-activated forms of the nine-carbon nonulosonate (NulO) sugar N-acetyl-neuraminic Acid (Neu5Ac), a sialic Acid (Sia) abundant in humans. This allows evasion of complement-mediated killing by recruiting factor H (FH), an inhibitor of the alternative complement pathway, and by limiting classical pathway activation (“serum-resistance”). We utilized CMP salts of six additional natural or synthetic NulOs, Neu5Gc, Neu5Gc8Me, Neu5Ac9Ac, Neu5Ac9Az, legionaminic Acid (Leg5Ac7Ac) and Pseudaminic Acid (Pse5Ac7Ac), to define structural requirements of Sia-mediated serum-resistance. While all NulOs except Pse5Ac7Ac were incorporated into the LNnT-LOS, only Neu5Gc incorporation yielded high-level serum-resistance and FH binding that was comparable to Neu5Ac, whereas Neu5Ac9Az and Leg5Ac7Ac incorporation left bacteria fully serum-sensitive and did not enhance FH binding. Neu5Ac9Ac and Neu5Gc8Me rendered bacteria resistant only to low serum concentrations. While serum-resistance mediated by Neu5Ac was associated with classical pathway inhibition (decreased IgG binding and C4 deposition), Leg5Ac7Ac and Neu5Ac9Az incorporation did not inhibit the classical pathway. Remarkably, CMP-Neu5Ac9Az and CMP-Leg5Ac7Ac each prevented serum-resistance despite a 100-fold molar excess of CMP-Neu5Ac in growth media. The concomitant presence of Leg5Ac7Ac and Neu5Ac on LOS resulted in uninhibited classical pathway activation. Surprisingly, despite near-maximal FH binding in this instance, the alternative pathway was not regulated and factor Bb remained associated with bacteria. Intravaginal administration of CMP-Leg5Ac7Ac to BALB/c mice infected with gonorrhea (including a multidrug-resistant isolate) reduced clearance times and infection burden. Bacteria recovered from CMP-Leg5Ac7Ac-treated mice were sensitive to human complement ex vivo, simulating in vitro findings. These data reveal critical roles for the Sia exocyclic side-chain in gonococcal serum-resistance. Such CMP-NulO analogs may provide a novel therapeutic strategy against the global threat of multidrug-resistant gonorrhea.

  • small molecule inhibitors of the Pseudaminic Acid biosynthetic pathway targeting motility as a key bacterial virulence factor
    Antimicrobial Agents and Chemotherapy, 2014
    Co-Authors: Robert Menard, Annie Aubry, Ian C. Schoenhofen, Patrice Bouchard, Christopher W Reid, Paule Lachance, Susan M Twine, Kelly M Fulton, Herve Hogues, Enrico O Purisima
    Abstract:

    Helicobacter pylori is motile by means of polar flagella, and this motility has been shown to play a critical role in pathogenicity. The major structural flagellin proteins have been shown to be glycosylated with the nonulosonate sugar, Pseudaminic Acid (Pse). This glycan is unique to microorganisms, and the process of flagellin glycosylation is required for H. pylori flagellar assembly and consequent motility. As such, the Pse biosynthetic pathway offers considerable potential as an antivirulence drug target, especially since motility is required for H. pylori colonization and persistence in the host. This report describes screening the five Pse biosynthetic enzymes for small-molecule inhibitors using both high-throughput screening (HTS) and in silico (virtual screening [VS]) approaches. Using a 100,000-compound library, 1,773 hits that exhibited a 40% threshold inhibition at a 10 μM concentration were identified by HTS. In addition, VS efforts using a 1.6-million compound library directed at two pathway enzymes identified 80 hits, 4 of which exhibited reasonable inhibition at a 10 μM concentration in vitro. Further secondary screening which identified 320 unique molecular structures or validated hits was performed. Following kinetic studies and structure-activity relationship (SAR) analysis of selected inhibitors from our refined list of 320 compounds, we demonstrated that three inhibitors with 50% inhibitory concentrations (IC50s) of approximately 14 μM, which belonged to a distinct chemical cluster, were able to penetrate the Gram-negative cell membrane and prevent formation of flagella.

  • structural and functional analysis of campylobacter jejuni pseg a udp sugar hydrolase from the Pseudaminic Acid biosynthetic pathway
    Journal of Biological Chemistry, 2009
    Co-Authors: Erumbi S Rangarajan, Susan M. Logan, Dennis M. Whitfield, A Proteau, Zhanna Potetinova, Enrico O Purisima, Miroslaw Cygler, A Matte, Traian Sulea, Ian C. Schoenhofen
    Abstract:

    Flagella of the bacteria Helicobacter pylori and Campylobacter jejuni are important virulence determinants, whose proper assembly and function are dependent upon glycosylation at multiple positions by sialic Acid-like sugars, such as 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-nonulosonic Acid (Pseudaminic Acid (Pse)). The fourth enzymatic step in the Pseudaminic Acid pathway, the hydrolysis of UDP-2,4-diacetamido-2,4,6-trideoxy-β-l-altropyranose to generate 2,4-diacetamido-2,4,6-trideoxy-l-altropyranose, is performed by the nucleotide sugar hydrolase PseG. To better understand the molecular basis of the PseG catalytic reaction, we have determined the crystal structures of C. jejuni PseG in apo-form and as a complex with its UDP product at 1.8 and 1.85 Å resolution, respectively. In addition, molecular modeling was utilized to provide insight into the structure of the PseG-substrate complex. This modeling identifies a His17-coordinated water molecule as the putative nucleophile and suggests the UDP-sugar substrate adopts a twist-boat conformation upon binding to PseG, enhancing the exposure of the anomeric bond cleaved and favoring inversion at C-1. Furthermore, based on these structures a series of amino Acid substitution derivatives were constructed, altering residues within the active site, and each was kinetically characterized to examine its contribution to PseG catalysis. In conjunction with structural comparisons, the almost complete inactivation of the PseG H17F and H17L derivatives suggests that His17 functions as an active site base, thereby activating the nucleophilic water molecule for attack of the anomeric C–O bond of the UDP-sugar. As the PseG structure reveals similarity to those of glycosyltransferase family-28 members, in particular that of Escherichia coli MurG, these findings may also be of relevance for the mechanistic understanding of this important enzyme family.

  • The Engineering of Bacteria Bearing Azido-Pseudaminic Acid-Modified Flagella
    ChemBioChem, 2009
    Co-Authors: Annie Aubry, Ian C. Schoenhofen, Susan M. Logan, Martin E Tanner
    Abstract:

    Catch a tiger by the tail: We have demonstrated that by feeding nonmotile mutant C. jejuni bacteria with a neutral azide-labelled Pseudaminic Acid precursor we can restore their ability to generate functional flagella. The presence of azido-Pseudaminic Acid on the surface of the flagella provides a bio-orthogonal chemical handle that can be used to modify the flagellar proteins.

  • mechanistic studies on pseb of Pseudaminic Acid biosynthesis a udp n acetylglucosamine 5 inverting 4 6 dehydratase
    Bioorganic Chemistry, 2008
    Co-Authors: James P Morrison, Ian C. Schoenhofen, Martin E Tanner
    Abstract:

    UDP-N-acetylglucosamine 5-inverting 4,6-dehydratase (PseB) is a unique sugar nucleotide dehydratase that inverts the C-5″ stereocentre during conversion of UDP-N-acetylglucosamine to UDP-2-acetamido-2,6-dideoxy-β-l-arabino-hexos-4-ulose. PseB catalyzes the first step in the biosynthesis of Pseudaminic Acid, which is found as a post-translational modification on the flagellin of Campylobacter jejuni and Helicobacter pylori. PseB is proposed to use its tightly bound NADP+ to oxidize UDP-GlcNAc at C-4″, enabling dehydration. The α,β unsaturated ketone intermediate is then reduced by delivery of the hydride to C-6″ and a proton to C-5″. Consistent with this, PseB from C. jejuni has been found to incorporate deuterium into the C-5″ position of product during catalysis in D2O. Likewise, PseB catalyzes solvent isotope exchange into the H-5″ position of product, and eliminates HF from the alternate substrate, UDP-6-deoxy-6-fluoro-GlcNAc. Mutants of the putative catalytic residues aspartate 126, lysine 127 and tyrosine 135 have severely compromised dehydratase, solvent isotope exchange, and HF elimination activities.

Martin E Tanner - One of the best experts on this subject based on the ideXlab platform.

  • The Engineering of Bacteria Bearing Azido-Pseudaminic Acid-Modified Flagella
    ChemBioChem, 2009
    Co-Authors: Annie Aubry, Ian C. Schoenhofen, Susan M. Logan, Martin E Tanner
    Abstract:

    Catch a tiger by the tail: We have demonstrated that by feeding nonmotile mutant C. jejuni bacteria with a neutral azide-labelled Pseudaminic Acid precursor we can restore their ability to generate functional flagella. The presence of azido-Pseudaminic Acid on the surface of the flagella provides a bio-orthogonal chemical handle that can be used to modify the flagellar proteins.

  • mechanistic studies on pseb of Pseudaminic Acid biosynthesis a udp n acetylglucosamine 5 inverting 4 6 dehydratase
    Bioorganic Chemistry, 2008
    Co-Authors: James P Morrison, Ian C. Schoenhofen, Martin E Tanner
    Abstract:

    UDP-N-acetylglucosamine 5-inverting 4,6-dehydratase (PseB) is a unique sugar nucleotide dehydratase that inverts the C-5″ stereocentre during conversion of UDP-N-acetylglucosamine to UDP-2-acetamido-2,6-dideoxy-β-l-arabino-hexos-4-ulose. PseB catalyzes the first step in the biosynthesis of Pseudaminic Acid, which is found as a post-translational modification on the flagellin of Campylobacter jejuni and Helicobacter pylori. PseB is proposed to use its tightly bound NADP+ to oxidize UDP-GlcNAc at C-4″, enabling dehydration. The α,β unsaturated ketone intermediate is then reduced by delivery of the hydride to C-6″ and a proton to C-5″. Consistent with this, PseB from C. jejuni has been found to incorporate deuterium into the C-5″ position of product during catalysis in D2O. Likewise, PseB catalyzes solvent isotope exchange into the H-5″ position of product, and eliminates HF from the alternate substrate, UDP-6-deoxy-6-fluoro-GlcNAc. Mutants of the putative catalytic residues aspartate 126, lysine 127 and tyrosine 135 have severely compromised dehydratase, solvent isotope exchange, and HF elimination activities.

  • Biosynthesis of CMP-N,N'-diacetyllegionaminic Acid from UDP-N,N'-diacetylbacillosamine in Legionella pneumophila.
    Biochemistry, 2008
    Co-Authors: Pavel A. Glaze, N. Martin Young, David C. Watson, Martin E Tanner
    Abstract:

    Legionaminic Acid is a nine-carbon α-keto Acid that is similar in structure to other members of the sialic Acid family that includes neuraminic Acid and Pseudaminic Acid. It is found as a component of the lipopolysaccharide in several bacterial species and is perhaps best known for its presence in the 0-antigen of the causative agent of Legionnaires' disease, Legionella pneumophila. In this work, the enzymes responsible for the biosynthesis and activation of N,N'-diacetyllegionaminic Acid are identified for the first time. A cluster of three L. pneumophila genes bearing homology to known sialic Acid biosynthetic genes (neuA,B, C) were cloned and overexpressed in Escherichia coli. The NeuC homologue was found to be a hydrolyzing UDP-N,N'-diacetylbacillosamine 2-epimerase that converts UDP-N,N'-diacetylbacillosamine into 2,4-diacetamido-2,4,6-trideoxymannose and UDP. Stereochemical and isotopic labeling studies showed that the enzyme utilizes a mechanism involving an initial anti elimination of UDP to form a glycal intermediate and a subsequent syn addition of water to generate product. This is similar to the hydrolyzing UDP-N-acetylglucosamine 2-epimerase (NeuC) of sialic Acid biosynthesis, but the L. pneumophila enzyme would not accept UDP-GlcNAc as an alternate substrate. The NeuB homologue was found to be a N,N'-diacetyllegionaminic Acid synthase that condenses 2,4-diacetamido-2,4,6-trideoxymannose with phosphoenolpyruvate (PEP), although the in vitro activity of the recombinant enzyme (isolated as a MalE fusion protein) was very low. The synthase activity was dependent on the presence of a divalent metal ion, and the reaction proceeded via a C-O bond cleavage process, similar to the reactions catalyzed by the sialic Acid and Pseudaminic Acid synthases. Finally, the NeuA homologue was shown to possess the CMP-N,N'-diacetyllegionaminic Acid synthetase activity that generates the activated form of legionaminic Acid used in lipopolysaccharide biosynthesis. Together, the three enzymes constitute a pathway that converts a UDP-linked bacillosamine derivative into a CMP-linked legionaminic Acid derivative.

  • pseg of Pseudaminic Acid biosynthesis a udp sugar hydrolase as a masked glycosyltransferase
    Journal of Biological Chemistry, 2006
    Co-Authors: Martin E Tanner
    Abstract:

    Abstract The flagellin proteins in pathogenic bacteria such as Campylobacter jejuni and Helicobacter pylori are heavily glycosylated with the nine-carbon α-keto Acid, Pseudaminic Acid. The presence of this posttranslational modification is absolutely required for assembly of functional flagella. Since motility is required for colonization, Pseudaminic Acid biosynthesis represents a virulence factor in these bacteria. Pseudaminic Acid is generated from UDP-N-acetylglucosamine in five biosynthetic steps. The final step has been shown to involve the condensation of 2,4-diacetamido-2,4,6-trideoxy-l-altrose (6-deoxy-Altdi-NAc) with phosphoenolpyruvate as catalyzed by the enzyme Pseudaminic Acid synthase, NeuB3. The 6-deoxy-AltdiNAc used in this process is generated from its nucleotide-linked form, UDP-6-deoxy-AltdiNAc, by the action of a hydrolase that cleaves the glycosidic bond and releases UDP. This manuscript describes the first characterization of a UDP-6-deoxy-AltdiNAc hydrolase, namely PseG (Cj1312) from C. jejuni. The activity of this enzyme is independent of the presence of divalent metal ions, and the values of the catalytic constants were found to be kcat = 27 s–1 and Km = 174 μm. The enzyme was shown to hydrolyze the substrate with an overall inversion of stereochemistry at C-1 and to utilize a C–O bond cleavage mechanism during catalysis. These results, coupled with homology comparisons, suggest that the closest ancestors to the hydrolase are members of the metal-independent GT-B family of glycosyltransferases that include the enzyme MurG.

  • identification and characterization of neub3 from campylobacter jejuni as a Pseudaminic Acid synthase
    Journal of Biological Chemistry, 2005
    Co-Authors: Wayne K Chou, Scott Dick, Warren W Wakarchuk, Martin E Tanner
    Abstract:

    Abstract Campylobacter jejuni and Campylobacter coli are the main causes of bacterial diarrhea worldwide, and Helicobacter pylori is known to cause duodenal ulcers. In all of these pathogenic organisms, the flagellin proteins are heavily glycosylated with a 2-keto-3-deoxy Acid, Pseudaminic Acid (5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-nonulosonic Acid). The presence of Pseudaminic Acid is required for the proper development of the flagella and is thereby necessary for motility in, and invasion of, the host. In this study we report the first characterization of NeuB3 from C. jejuni as a Pseudaminic Acid synthase; the enzyme directly responsible for the biosynthesis of Pseudaminic Acid. Pseudaminic Acid synthase catalyzes the condensation of phosphoenolpyruvate (PEP) with the hexose, 2,4-diacetamido-2,4,6-trideoxy-l-altrose (6-deoxy-AltdiNAc), to form Pseudaminic Acid and phosphate. The enzymatic activity was monitored using 1H and 31P NMR spectroscopy, and the product was isolated and characterized. Kinetic analysis reveals that Pseudaminic Acid synthase requires the presence of a divalent metal ion for catalysis and that optimal catalysis occurs at pH 7.0. A coupled enzymatic assay gave the values for kcat of 0.65 ± 0.01 s–1, KmPEP of 6.5 ± 0.4 μm, and Km6-deoxy-AltdiNAc of 9.5 ± 0.7 μm. A mechanistic study on Pseudaminic Acid synthase, using [2-18O]PEP, shows that catalysis proceeds through a C-O bond cleavage mechanism similar to other PEP condensing synthases such as sialic Acid synthase.

Jean-robert Brisson - One of the best experts on this subject based on the ideXlab platform.

  • CMP‐Pseudaminic Acid is a Natural Potent Inhibitor of PseB, the First Enzyme of the Pseudaminic Acid Pathway in Campylobacter jejuni and Helicobacter pylori
    ChemMedChem, 2008
    Co-Authors: David J. Mcnally, Ian C. Schoenhofen, Susan M. Logan, R. Scott Houliston, Nam H. Khieu, Dennis M. Whitfield, Harold C. Jarrell, Jean-robert Brisson
    Abstract:

    Deadly decorations. Campylobacter jejuni and Helicobacter pylori decorate their flagella, which are essential for virulence, with Pseudaminic Acid (Pse). Pse production is feedback-regulated in the bacterial cell by CMP-Pseudaminic Acid, a potent inhibitor of PseB, the first enzyme of the Pse pathway. Herein, STD NMR was used to map binding epitopes for PseB and to characterize the interaction between PseB and CMP-Pse.Peer reviewed: YesNRC publication: Ye

  • Cover Picture: CMP-Pseudaminic Acid is a Natural Potent Inhibitor of PseB, the First Enzyme of the Pseudaminic Acid Pathway in Campylobacter jejuni and Helicobacter pylori (ChemMedChem 1/2008)
    ChemMedChem, 2008
    Co-Authors: David J. Mcnally, Ian C. Schoenhofen, Susan M. Logan, R. Scott Houliston, Nam H. Khieu, Dennis M. Whitfield, Harold C. Jarrell, Jean-robert Brisson
    Abstract:

    The cover picture shows the six enzymes (Pse B, C, H, G, I, F) responsible for producing CMP-Pseudaminic Acid (CMP-Pse) starting from UDP-GlcNAc in Campylobacter jejuni and Helicobacter pylori. These pathogens modify their flagella with sialic Acid-like sugars such as Pseudaminic Acid (Pse) which are required for flagellar assembly, motility, and hence virulence. Pse B plays a central role in Pse biosynthesis and is also thought to be implicated in other glycan pathways making it a prime therapeutic target. Saturation transfer difference nuclear magnetic resonance spectroscopy (STD NMR) was used to determine binding epitopes for Pse B and to characterize Pse B inhibition with CMP-Pse at the molecular level. Docking studies and CORCEMA calculations validated STD NMR results and revealed that CMP-Pse and UDP-GlcNAc adopt similar conformations within the Pse B active site. These findings will guide the development of small-molecule inhibitors as a means to pharmaceutically control C. jejuni and H. pylori infections. For details, see the Communication by D. J. McNally, et al. on p. 55 ff.Peer reviewed: YesNRC publication: Ye

  • Targeted metabolomics analysis of Campylobacter coli VC167 reveals legionaminic Acid derivatives as novel flagellar glycans.
    Journal of Biological Chemistry, 2007
    Co-Authors: David J. Mcnally, Annie Aubry, Nam H. Khieu, Dennis M. Whitfield, Jean-robert Brisson, Cheryl P Ewing, Patricia Guerry, Susan M. Logan
    Abstract:

    Abstract Glycosylation of Campylobacter flagellin is required for the biogenesis of a functional flagella filament. Recently, we used a targeted metabolomics approach using mass spectrometry and NMR to identify changes in the metabolic profile of wild type and mutants in the flagellar glycosylation locus, characterize novel metabolites, and assign function to genes to define the Pseudaminic Acid biosynthetic pathway in Campylobacter jejuni 81-176 (McNally, D. J., Hui, J. P., Aubry, A. J., Mui, K. K., Guerry, P., Brisson, J. R., Logan, S. M., and Soo, E. C. (2006) J. Biol. Chem. 281, 18489-18498). In this study, we use a similar approach to further define the glycome and metabolomic complement of nucleotide-activated sugars in Campylobacter coli VC167. Herein we demonstrate that, in addition to CMP-Pseudaminic Acid, C. coli VC167 also produces two structurally distinct nucleotide-activated nonulosonate sugars that were observed as negative ions at m/z 637 and m/z 651 (CMP-315 and CMP-329). Hydrophilic interaction liquid chromatography-mass spectrometry yielded suitable amounts of the pure sugar nucleotides for NMR spectroscopy using a cold probe. Structural analysis in conjunction with molecular modeling identified the sugar moieties as acetamidino and N-methylacetimidoyl derivatives of legionaminic Acid (Leg5Am7Ac and Leg5AmNMe7Ac). Targeted metabolomic analyses of isogenic mutants established a role for the ptmA-F genes and defined two new ptm genes in this locus as legionaminic Acid biosynthetic enzymes. This is the first report of legionaminic Acid in Campylobacter sp. and the first report of legionaminic Acid derivatives as modifications on a protein.

  • Identification of Labile UDP-Ketosugars in Helicobacter pylori, Campylobacter jejuni and Pseudomonas aeruginosa: Key Metabolites used to make Glycan Virulence Factors
    ChemBioChem, 2006
    Co-Authors: David J. Mcnally, Ian C. Schoenhofen, Susan M. Logan, Dennis M. Whitfield, Evgeny Vinogradov, Erin F. Mulrooney, Jean-robert Brisson
    Abstract:

    Infectious microorganisms pose an ongoing threat to the health of humans through the development of resistance to antibiotics. Consequently, there is a continual need for novel antimicrobials. Several bacteria produce glycan structures which are attractive therapeutic targets as many are virulence factors that are unique to prokaryotes. Examples can be found in the surface glycans produced by the Gram-negative bacteria Pseudomonas aeruginosa, an opportunistic pathogen that infects individuals whose defenses are compromised, Campylobacter jejuni, a principal cause of acute gastroenteritis, and Helicobacter pylori, a major etiological agent of gastroduodenal disease and the only bacterium to be associated with cancer. Lipopolysaccharides (LPS) produced by many serotypes of P. aeruginosa contain b-l-FucNAc, a deoxyaminohexose that is also a constituent of LPS in Escherichia coli and the capsular polysaccharide (CPS) in Staphylococcus aureus and Streptococcus pneumoniae. C. jejuni is unique amongst bacteria as it generally modifies over 30 proteins with an N-linked heptasaccharide that contains the bacillosamine derivative 2,4-diacetamido-2,4,6-trideoxy-a-d-Glc (a-d-QuiNAc4NAc). H. pylori and C. jejuni both decorate their flagella with the sialic Acid-like sugar 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-a-l-mannononulosonic Acid or Pseudaminic Acid (Pse). 7] Pse appears to be necessary for flagellar assembly because mutants deficient in the ability to make this sugar lack flagella and have non-

  • elucidation of the cmp Pseudaminic Acid pathway in helicobacter pylori synthesis from udp n acetylglucosamine by a single enzymatic reaction
    Glycobiology, 2006
    Co-Authors: Ian C. Schoenhofen, David J. Mcnally, Jean-robert Brisson, Susan M. Logan
    Abstract:

    Flagellin glycosylation is a necessary modification allowing flagellar assembly, bacterial motility, colonization, and hence virulence for the gastrointestinal pathogen Helicobacter pylori [Josenhans, C., Vossebein, L., Friedrich, S., and Suerbaum, S. (2002) FEMS Microbiol. Lett., 210, 165-172; Schirm, M., Schoenhofen, I.C., Logan, S.M., Waldron, K.C., and Thibault, P. (2005) Anal. Chem., 77, 7774-7782]. A causative agent of gastric and duodenal ulcers, H. pylori, heavily modifies its flagellin with the sialic Acid-like sugar 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-alpha-l-manno-nonulosonic Acid (Pseudaminic Acid). Because this sugar is unique to bacteria, its biosynthetic pathway offers potential as a novel therapeutic target. We have identified six H. pylori enzymes, which reconstitute the complete biosynthesis of Pseudaminic Acid, and its nucleotide-activated form CMP-Pseudaminic Acid, from UDP-N-acetylglucosamine (UDP-GlcNAc). The pathway intermediates and final product were identified from monitoring sequential reactions with nuclear magnetic resonance (NMR) spectroscopy, thereby confirming the function of each biosynthetic enzyme. Remarkably, the conversion of UDP-GlcNAc to CMP-Pseudaminic Acid was achieved in a single reaction combining six enzymes. This represents the first complete in vitro enzymatic synthesis of a sialic Acid-like sugar and sets the groundwork for future small molecule inhibitor screening and design. Moreover, this study provides a strategy for efficient large-scale synthesis of novel medically relevant bacterial sugars that has not been attainable by chemical methods alone.

David J. Mcnally - One of the best experts on this subject based on the ideXlab platform.

  • CMP‐Pseudaminic Acid is a Natural Potent Inhibitor of PseB, the First Enzyme of the Pseudaminic Acid Pathway in Campylobacter jejuni and Helicobacter pylori
    ChemMedChem, 2008
    Co-Authors: David J. Mcnally, Ian C. Schoenhofen, Susan M. Logan, R. Scott Houliston, Nam H. Khieu, Dennis M. Whitfield, Harold C. Jarrell, Jean-robert Brisson
    Abstract:

    Deadly decorations. Campylobacter jejuni and Helicobacter pylori decorate their flagella, which are essential for virulence, with Pseudaminic Acid (Pse). Pse production is feedback-regulated in the bacterial cell by CMP-Pseudaminic Acid, a potent inhibitor of PseB, the first enzyme of the Pse pathway. Herein, STD NMR was used to map binding epitopes for PseB and to characterize the interaction between PseB and CMP-Pse.Peer reviewed: YesNRC publication: Ye

  • Cover Picture: CMP-Pseudaminic Acid is a Natural Potent Inhibitor of PseB, the First Enzyme of the Pseudaminic Acid Pathway in Campylobacter jejuni and Helicobacter pylori (ChemMedChem 1/2008)
    ChemMedChem, 2008
    Co-Authors: David J. Mcnally, Ian C. Schoenhofen, Susan M. Logan, R. Scott Houliston, Nam H. Khieu, Dennis M. Whitfield, Harold C. Jarrell, Jean-robert Brisson
    Abstract:

    The cover picture shows the six enzymes (Pse B, C, H, G, I, F) responsible for producing CMP-Pseudaminic Acid (CMP-Pse) starting from UDP-GlcNAc in Campylobacter jejuni and Helicobacter pylori. These pathogens modify their flagella with sialic Acid-like sugars such as Pseudaminic Acid (Pse) which are required for flagellar assembly, motility, and hence virulence. Pse B plays a central role in Pse biosynthesis and is also thought to be implicated in other glycan pathways making it a prime therapeutic target. Saturation transfer difference nuclear magnetic resonance spectroscopy (STD NMR) was used to determine binding epitopes for Pse B and to characterize Pse B inhibition with CMP-Pse at the molecular level. Docking studies and CORCEMA calculations validated STD NMR results and revealed that CMP-Pse and UDP-GlcNAc adopt similar conformations within the Pse B active site. These findings will guide the development of small-molecule inhibitors as a means to pharmaceutically control C. jejuni and H. pylori infections. For details, see the Communication by D. J. McNally, et al. on p. 55 ff.Peer reviewed: YesNRC publication: Ye

  • Targeted metabolomics analysis of Campylobacter coli VC167 reveals legionaminic Acid derivatives as novel flagellar glycans.
    Journal of Biological Chemistry, 2007
    Co-Authors: David J. Mcnally, Annie Aubry, Nam H. Khieu, Dennis M. Whitfield, Jean-robert Brisson, Cheryl P Ewing, Patricia Guerry, Susan M. Logan
    Abstract:

    Abstract Glycosylation of Campylobacter flagellin is required for the biogenesis of a functional flagella filament. Recently, we used a targeted metabolomics approach using mass spectrometry and NMR to identify changes in the metabolic profile of wild type and mutants in the flagellar glycosylation locus, characterize novel metabolites, and assign function to genes to define the Pseudaminic Acid biosynthetic pathway in Campylobacter jejuni 81-176 (McNally, D. J., Hui, J. P., Aubry, A. J., Mui, K. K., Guerry, P., Brisson, J. R., Logan, S. M., and Soo, E. C. (2006) J. Biol. Chem. 281, 18489-18498). In this study, we use a similar approach to further define the glycome and metabolomic complement of nucleotide-activated sugars in Campylobacter coli VC167. Herein we demonstrate that, in addition to CMP-Pseudaminic Acid, C. coli VC167 also produces two structurally distinct nucleotide-activated nonulosonate sugars that were observed as negative ions at m/z 637 and m/z 651 (CMP-315 and CMP-329). Hydrophilic interaction liquid chromatography-mass spectrometry yielded suitable amounts of the pure sugar nucleotides for NMR spectroscopy using a cold probe. Structural analysis in conjunction with molecular modeling identified the sugar moieties as acetamidino and N-methylacetimidoyl derivatives of legionaminic Acid (Leg5Am7Ac and Leg5AmNMe7Ac). Targeted metabolomic analyses of isogenic mutants established a role for the ptmA-F genes and defined two new ptm genes in this locus as legionaminic Acid biosynthetic enzymes. This is the first report of legionaminic Acid in Campylobacter sp. and the first report of legionaminic Acid derivatives as modifications on a protein.

  • Identification of Labile UDP-Ketosugars in Helicobacter pylori, Campylobacter jejuni and Pseudomonas aeruginosa: Key Metabolites used to make Glycan Virulence Factors
    ChemBioChem, 2006
    Co-Authors: David J. Mcnally, Ian C. Schoenhofen, Susan M. Logan, Dennis M. Whitfield, Evgeny Vinogradov, Erin F. Mulrooney, Jean-robert Brisson
    Abstract:

    Infectious microorganisms pose an ongoing threat to the health of humans through the development of resistance to antibiotics. Consequently, there is a continual need for novel antimicrobials. Several bacteria produce glycan structures which are attractive therapeutic targets as many are virulence factors that are unique to prokaryotes. Examples can be found in the surface glycans produced by the Gram-negative bacteria Pseudomonas aeruginosa, an opportunistic pathogen that infects individuals whose defenses are compromised, Campylobacter jejuni, a principal cause of acute gastroenteritis, and Helicobacter pylori, a major etiological agent of gastroduodenal disease and the only bacterium to be associated with cancer. Lipopolysaccharides (LPS) produced by many serotypes of P. aeruginosa contain b-l-FucNAc, a deoxyaminohexose that is also a constituent of LPS in Escherichia coli and the capsular polysaccharide (CPS) in Staphylococcus aureus and Streptococcus pneumoniae. C. jejuni is unique amongst bacteria as it generally modifies over 30 proteins with an N-linked heptasaccharide that contains the bacillosamine derivative 2,4-diacetamido-2,4,6-trideoxy-a-d-Glc (a-d-QuiNAc4NAc). H. pylori and C. jejuni both decorate their flagella with the sialic Acid-like sugar 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-a-l-mannononulosonic Acid or Pseudaminic Acid (Pse). 7] Pse appears to be necessary for flagellar assembly because mutants deficient in the ability to make this sugar lack flagella and have non-

  • elucidation of the cmp Pseudaminic Acid pathway in helicobacter pylori synthesis from udp n acetylglucosamine by a single enzymatic reaction
    Glycobiology, 2006
    Co-Authors: Ian C. Schoenhofen, David J. Mcnally, Jean-robert Brisson, Susan M. Logan
    Abstract:

    Flagellin glycosylation is a necessary modification allowing flagellar assembly, bacterial motility, colonization, and hence virulence for the gastrointestinal pathogen Helicobacter pylori [Josenhans, C., Vossebein, L., Friedrich, S., and Suerbaum, S. (2002) FEMS Microbiol. Lett., 210, 165-172; Schirm, M., Schoenhofen, I.C., Logan, S.M., Waldron, K.C., and Thibault, P. (2005) Anal. Chem., 77, 7774-7782]. A causative agent of gastric and duodenal ulcers, H. pylori, heavily modifies its flagellin with the sialic Acid-like sugar 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-alpha-l-manno-nonulosonic Acid (Pseudaminic Acid). Because this sugar is unique to bacteria, its biosynthetic pathway offers potential as a novel therapeutic target. We have identified six H. pylori enzymes, which reconstitute the complete biosynthesis of Pseudaminic Acid, and its nucleotide-activated form CMP-Pseudaminic Acid, from UDP-N-acetylglucosamine (UDP-GlcNAc). The pathway intermediates and final product were identified from monitoring sequential reactions with nuclear magnetic resonance (NMR) spectroscopy, thereby confirming the function of each biosynthetic enzyme. Remarkably, the conversion of UDP-GlcNAc to CMP-Pseudaminic Acid was achieved in a single reaction combining six enzymes. This represents the first complete in vitro enzymatic synthesis of a sialic Acid-like sugar and sets the groundwork for future small molecule inhibitor screening and design. Moreover, this study provides a strategy for efficient large-scale synthesis of novel medically relevant bacterial sugars that has not been attainable by chemical methods alone.