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Paul Kosma - One of the best experts on this subject based on the ideXlab platform.
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adp Heptose a novel pathogen associated molecular pattern identified in helicobacter pylori
The FASEB Journal, 2019Co-Authors: Lennart Pfannkuch, Robert Hurwitz, Jan Traulsen, Janine Sigulla, Marcella Poeschke, Laura Matzner, Paul Kosma, Monika Schmid, Thomas F. MeyerAbstract:The gastric pathogen Helicobacter pylori activates the NF-κB pathway in human epithelial cells via the recently discovered α-kinase 1 TRAF-interacting protein with forkhead-associated domain (TIFA) axis. We and others showed that this pathway can be triggered by Heptose 1,7-bisphosphate (HBP), an LPS intermediate produced in gram-negative bacteria that represents a new pathogen-associated molecular pattern (PAMP). Here, we report that our attempts to identify HBP in lysates of H. pylori revealed surprisingly low amounts, failing to explain NF-κB activation. Instead, we identified ADP-glycero-β-D-manno-Heptose (ADP Heptose), a derivative of HBP, as the predominant PAMP in lysates of H. pylori and other gram-negative bacteria. ADP Heptose exhibits significantly higher activity than HBP, and cells specifically sensed the presence of the β-form, even when the compound was added extracellularly. The data lead us to conclude that ADP Heptose not only constitutes the key PAMP responsible for H. pylori-induced NF-κB activation in epithelial cells, but it acts as a general gram-negative bacterial PAMP.-Pfannkuch, L., Hurwitz, R., Traulsen, J., Sigulla, J., Poeschke, M., Matzner, L., Kosma, P., Schmid, M., Meyer, T. F. ADP Heptose, a novel pathogen-associated molecular pattern identified in Helicobacter pylori.
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adp Heptose a novel pathogen associated molecular pattern associated with helicobacter pylori type 4 secretion
bioRxiv, 2018Co-Authors: Lennart Pfannkuch, Robert Hurwitz, Jan Traulsen, Paul Kosma, Monika Schmid, Thomas F. MeyerAbstract:The gastric pathogen Helicobacter pylori activates the NF-kappaB pathway in human epithelial cells via the alpha-kinase 1 (Alpk1) TIFA axis. We and others have previously shown that Heptose 1,7-bisphosphate (HBP) acts as a pathogen-associated molecular pattern (PAMP). HBP is an intermediate of lipopolysaccharide (LPS) synthesis in H. pylori and other gram-negative bacteria. Deletion of the hldE (rfaE) gene encoding the enzyme responsible for HBP synthesis, as well as deletion of further upstream genes, causes loss of NF-κB stimulation by H. pylori, while deletion of the downstream phosphatase encoding gene gmhB does not. This has led to the conclusion that HBP is the PAMP responsible for NF-κB induction. Here, our attempts to identify HBP in lysates of H. pylori revealed surprisingly low amounts that fail to explain NF-κB activation. Instead, we identified ADP Heptose, a major downstream metabolite of HdlE, as the predominant PAMP in H. pylori lysates, exhibiting around 100-fold stronger activity compared to HBP. It therefore appears that synthesis of ADP Heptose from HBP in H. pylori occurs independently of GmhB. The data lead us to conclude that ADP Heptose constitutes the key PAMP, secreted via the pathogens cagPAI encoded type 4 secretion (T4SS).
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chemical synthesis of the innate immune modulator bacterial d glycero β d manno Heptose 1 7 bisphosphate hbp
Tetrahedron Letters, 2017Co-Authors: Alessio Borio, Andreas Hofinger, Paul Kosma, Alla ZamyatinaAbstract:Abstract The bacterial metabolite and potent innate immune modulator d - glycero -β- d - manno -Heptose-1,7-bisphosphate (HBP) and its α-configured counterpart d - glycero -α- d - manno -Heptose-1,7-bisphosphate were synthesized via stereoselective anomeric phosphorylation of the peracetylated d , d -Heptose 7-dibenzylphosphate by exploiting different nucleophilicity of equatorial and axial lactols in the d - manno -series. We also report a novel approach for anomeric phosphorylation using modified Mitsunobu reaction conditions and provide the first full structural characterization of HBP. The first chemical synthesis of HBP offers access to an anomerically pure structurally defined probe for biological studies and to a lead compound operating as a powerful stimulator of intracellular signaling for possible therapeutic immunomodulation.
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Chemical synthesis of the core oligosaccharide of bacterial lipopolysaccharide
Microbial Glycobiology, 2010Co-Authors: Paul KosmaAbstract:Publisher Summary Several in-depth reviews on the synthesis of higher-carbon bacterial sugars and of oligosaccharide structures of the core region of bacterial lipopolysaccharides (LPSs) have been published covering important aspects of chemical synthesis, protecting group strategies and preparation of neoglycoconjugates. This chapter reviews major strategies toward the chemical synthesis of the core region of LPSs of Gram-negative bacteria illustrated by representative examples, including Pseudomonas aeruginosa, Neisseriaceae, Vibrio parahaemolyticus, and Haemophilus. The focus is on the synthesis of structural units and neoglycoconjugates of the Heptose- and 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo)-containing inner core region listed according to the corresponding bacterial species. Furthermore, the chapter discusses the synthesis of phosphate-substituted determinants as well as selected outer-core units. Synthetic work targeting larger LPS core fragments could be helpful in elucidating the physiological and immunochemical role of phosphate substituents in the Heptose region.
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Synthesis of a deoxy analogue of ADP L-glycero-D-manno-Heptose
Carbohydrate research, 2007Co-Authors: Edit Balla, Alla Zamyatina, Andreas Hofinger, Paul KosmaAbstract:Abstract Starting from l -lyxose, indium-mediated chain elongation with allyl bromide followed by acetylation and oxidative cleavage of the double bond and deprotection afforded 2-deoxy- l - galacto -Heptose as a 2-deoxy analogue of the bacterial carbohydrate l - glycero - d - manno -Heptose in good overall yield. For the synthesis of the ADP-activated derivative, the 2-deoxy-Heptose was O-acetylated and transformed into the anomeric bromide derivative, which was then converted into the acetylated heptopyranosyl phosphate by reaction with tetrabutylammonium phosphate. Deprotection and separation of the anomeric phosphates furnished 2-deoxy-β- l - galacto -heptopyranosyl phosphate. Coupling of the acetylated heptosyl phosphate with AMP morpholidate afforded the acetylated ADP derivative in good yield. Removal of the acetyl groups gave the target compound ADP 2-deoxy- l - galacto -heptopyranose, which may serve as substrate analogue of bacterial ADP heptosyl transferases for biochemical and crystallographic studies.
Alla Zamyatina - One of the best experts on this subject based on the ideXlab platform.
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adp Heptose is a newly identified pathogen associated molecular pattern of shigella flexneri
EMBO Reports, 2018Co-Authors: Anne-sophie Dangeard, Johan Cornil, Linda Thai, Héloïse Rytter, Diego Garciaweber, Alla ZamyatinaAbstract:Abstract During an infection, the detection of pathogens is mediated through the interactions between pathogen‐associated molecular patterns (PAMPs) and pathogen recognition receptors. β‐Heptose 1,7‐bisphosphate (βHBP), an intermediate of the lipopolysaccharide (LPS) biosynthesis pathway, was recently identified as a bacterial PAMP. It was reported that βHBP sensing leads to oligomerization of TIFA proteins, a mechanism controlling NF‐κB activation and pro‐inflammatory gene expression. Here, we compare the ability of chemically synthesized βHBP and Shigella flexneri lysate to induce TIFA oligomerization in epithelial cells. We find that, unlike bacterial lysate, βHBP fails to initiate rapid TIFA oligomerization. It only induces delayed signaling, suggesting that βHBP must be processed intracellularly to trigger inflammation. Gene deletion and complementation analysis of the LPS biosynthesis pathway revealed that ADP‐Heptose is the bacterial metabolite responsible for rapid TIFA oligomerization. ADP‐Heptose sensing occurs down to 10 −10 M. During S. flexneri infection, it results in cytokine production, a process dependent on the kinase ALPK1. Altogether, our results rule out a major role of βHBP in S. flexneri infection and identify ADP‐Heptose as a new bacterial PAMP.
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chemical synthesis of the innate immune modulator bacterial d glycero β d manno Heptose 1 7 bisphosphate hbp
Tetrahedron Letters, 2017Co-Authors: Alessio Borio, Andreas Hofinger, Paul Kosma, Alla ZamyatinaAbstract:Abstract The bacterial metabolite and potent innate immune modulator d - glycero -β- d - manno -Heptose-1,7-bisphosphate (HBP) and its α-configured counterpart d - glycero -α- d - manno -Heptose-1,7-bisphosphate were synthesized via stereoselective anomeric phosphorylation of the peracetylated d , d -Heptose 7-dibenzylphosphate by exploiting different nucleophilicity of equatorial and axial lactols in the d - manno -series. We also report a novel approach for anomeric phosphorylation using modified Mitsunobu reaction conditions and provide the first full structural characterization of HBP. The first chemical synthesis of HBP offers access to an anomerically pure structurally defined probe for biological studies and to a lead compound operating as a powerful stimulator of intracellular signaling for possible therapeutic immunomodulation.
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Synthesis of a deoxy analogue of ADP L-glycero-D-manno-Heptose
Carbohydrate research, 2007Co-Authors: Edit Balla, Alla Zamyatina, Andreas Hofinger, Paul KosmaAbstract:Abstract Starting from l -lyxose, indium-mediated chain elongation with allyl bromide followed by acetylation and oxidative cleavage of the double bond and deprotection afforded 2-deoxy- l - galacto -Heptose as a 2-deoxy analogue of the bacterial carbohydrate l - glycero - d - manno -Heptose in good overall yield. For the synthesis of the ADP-activated derivative, the 2-deoxy-Heptose was O-acetylated and transformed into the anomeric bromide derivative, which was then converted into the acetylated heptopyranosyl phosphate by reaction with tetrabutylammonium phosphate. Deprotection and separation of the anomeric phosphates furnished 2-deoxy-β- l - galacto -heptopyranosyl phosphate. Coupling of the acetylated heptosyl phosphate with AMP morpholidate afforded the acetylated ADP derivative in good yield. Removal of the acetyl groups gave the target compound ADP 2-deoxy- l - galacto -heptopyranose, which may serve as substrate analogue of bacterial ADP heptosyl transferases for biochemical and crystallographic studies.
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Synthesis of C-glycosidically linked ADP glycero-β-d-manno-Heptose analogues
Tetrahedron: Asymmetry, 2007Co-Authors: Andrea Graziani, Alla Zamyatina, Hassan Amer, Andreas Hofinger, Paul KosmaAbstract:Abstract C-Glycosides of l - glycero - d - manno - and d - glycero - d - manno -Heptose containing either ( S )- or ( R )-2-hydroxypropyl aglycons are easily accessible compounds via condensation of reducing Heptoses with pentane-2,4-dione. 2′,3′-Di- O -acetyl adenosine was transformed into the corresponding 5′- O -cyanoethyl N , N -diisopropylaminophosphoramidite derivative, which was coupled in fair yields to the O-acetylated diastereoisomeric C-glycosidic alcohols. Oxidation of the phosphite triesters followed by deprotection furnished four ADP-Heptose analogues, wherein the heptosyl phosphate moiety had been replaced by a three carbon-skeleton. The compounds serving as substrate analogues will be used for co-crystallization experiments with ADP heptosyl transferases.
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biosynthesis pathway of adp l glycero β d manno Heptose in escherichia coli
Journal of Bacteriology, 2002Co-Authors: Bernd Kneidinger, Alla Zamyatina, Paul Kosma, Michael Graninger, Miguel A. Valvano, Cristina L Marolda, Fiona Mcarthur, Paul MessnerAbstract: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.
Stéphane P. Vincent - One of the best experts on this subject based on the ideXlab platform.
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synthesis of a biotinylated Heptose 1 7 bisphosphate analogue a probe to study immunity and inflammation
Organic and Biomolecular Chemistry, 2021Co-Authors: Lina Liang, Tong-you Wade Wei, Ming-daw Tsai, Jun Cao, Stéphane P. VincentAbstract:d-glycero-d-manno-Heptose-1β,7-bisphosphate (HBP) is a bacterial metabolite that can induce a TIFA-dependent innate immune response in mammals. It was recently discovered that after HBP enters into the cytoplasm of the host cell, it is transformed into ADP-Heptose-7-phosphate, which then leads to ALPK1-TIFA-dependent inflammatory response. In order to provide a molecular tool allowing the discovery of the proteins involved in this novel inflammatory pathway, we designed and synthesized a biotinylated analogue of HBP. This chemical probe displays an anomeric β-phosphate and a phosphonate at the 7-position, and a d-configured 6-position to which is attached the biotin moiety. To do so, different synthetic strategies were explored and described in this report. Moreover, we demonstrated that the biotinylated version of HBP is still biologically active and can activate the NF-κB pathway in HEK293T cells.
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Nonhydrolyzable Heptose Bis- and Monophosphate Analogues Modulate Pro-inflammatory TIFA-NF-κB Signaling.
Chembiochem : a European journal of chemical biology, 2020Co-Authors: Lina Liang, Tong-you Wade Wei, Wouter A. Herrebout, Ming-daw Tsai, Stéphane P. VincentAbstract:d-Glycero-d-manno-Heptose-1β,7-bisphosphate (HBP) and d-glycero-d-manno-Heptose-1β-phosphate (H1P) are bacterial metabolites that were recently shown to stimulate inflammatory responses in host cells through the activation of the TIFA-dependent NF-κB pathway. To better understand structure-based activity in relation to this process, a family of nonhydrolyzable phosphonate analogues of HBP and H1P was synthesized. The inflammation modulation by which these molecules induce the TIFA-NF-κB signal axis was evaluated in vivo at a low-nanomolar concentration (6 nM) and compared to that of the natural metabolites. Our data showed that three phosphonate analogues had similar stimulatory activity to HBP, whereas two phosphonates antagonized HBP-induced TIFA-NF-κB signaling. These results open new horizons for the design of pro-inflammatory and innate immune modulators that could be used as vaccine adjuvant.
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Synthesis of d-glycero-d-manno-Heptose 1,7-bisphosphate (HBP) featuring a β-stereoselective bis-phosphorylation
Tetrahedron Letters, 2017Co-Authors: Lina Liang, Stéphane P. VincentAbstract:Abstract d - glycero - d - manno -Heptose 1,7-bisphosphate (HBP) plays a unique role in bacteriology. We describe in this study a very efficient synthesis of HBP, featuring a highly 6-D-selective construction of the Heptose scaffold as well as a double phosphorylation step installing, in a single operation and in a β-stereoselective manner, the 1- and the 7-phosphates.
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Structure of the Escherichia coli Heptosyltransferase WaaC: Binary Complexes with ADP AND ADP-2-deoxy-2-fluoro Heptose
Journal of molecular biology, 2006Co-Authors: Sylvestre Grizot, François Moreau, Stéphane P. Vincent, Michèle Salem, Vanida Vongsouthi, Lionel Durand, Hirofumi Dohi, Sonia Escaich, Arnaud DucruixAbstract:Abstract 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 Kdo2-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.
Miguel A. Valvano - One of the best experts on this subject based on the ideXlab platform.
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Role of capsular modified Heptose in the virulence of Campylobacter jejuni
Molecular microbiology, 2015Co-Authors: Anthony Wong, Miguel A. Valvano, Dirk Lange, Sébastien Houle, Nikolay P. Arbatsky, Yuriy A. Knirel, Charles M. Dozois, Carole CreuzenetAbstract:The Campylobacter jejuni capsular polysaccharide is important for virulence and often contains a modified Heptose. In strain ATCC 700819 (a.k.a. NCTC 11168), the modified Heptose branches off from the capsular backbone and is directly exposed to the environment. We reported previously that the enzymes encoded by wcaG, mlghB and mlghC are involved in Heptose modification. Here, we show that inactivation of any of these genes leads to production of capsule lacking modified Heptose and alters the transcription of other capsule modification genes differentially. Inactivation of mlghB or mlghC, but not of wcaG, decreased susceptibility to bile salts and abrogated invasion of intestinal cells. All mutants showed increased sensitivity to serum killing, especially wcaG::cat, and had defects in colonization and persistence in chicken intestine, but did not show significant differences in adhesion, phagocytosis and intracellular survival in murine macrophages. Together, our findings suggest that the capsular Heptose modification pathway contributes to bacterial resistance against gastrointestinal host defenses and supports bacterial persistence via its role in serum resistance and invasion of intestinal cells. Our data further suggest a dynamic regulation of expression of this pathway in the gastrointestinal tract.
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Structural and Kinetic Characterization of the LPS Biosynthetic Enzyme D-alpha,beta-D-Heptose-1,7-bisphosphate Phosphatase (GmhB) from Escherichia coli
Biochemistry, 2010Co-Authors: Patricia L. Taylor, Miguel A. Valvano, Seiji Sugiman-marangos, Kun Zhang, Gerard D. Wright, Murray S. JunopAbstract:Lipopolysaccharide is a major component of the outer membrane of Gram-negative bacteria and provides a permeability barrier to many commonly used antibiotics. ADP-Heptose residues are an integral part of the LPS inner core, and mutants deficient in Heptose biosynthesis demonstrate increased membrane permeability. The Heptose biosynthesis pathway involves phosphorylation and dephosphorylation steps not found in other pathways for the synthesis of nucleotide sugar precursors. Consequently, the Heptose biosynthetic pathway has been marked as a novel target for antibiotic adjuvants, which are compounds that facilitate and potentiate antibiotic activity. D-{alpha},{beta}-D-Heptose-1,7-bisphosphate phosphatase (GmhB) catalyzes the third essential step of LPS Heptose biosynthesis. This study describes the first crystal structure of GmhB and enzymatic analysis of the protein. Structure-guided mutations followed by steady state kinetic analysis, together with established precedent for HAD phosphatases, suggest that GmhB functions through a phosphoaspartate intermediate. This study provides insight into the structure-function relationship of GmhB, a new target for combatting Gram-negative bacterial infection.
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structure and function of sedoheptulose 7 phosphate isomerase a critical enzyme for lipopolysaccharide biosynthesis and a target for antibiotic adjuvants
Journal of Biological Chemistry, 2008Co-Authors: Patricia L. Taylor, Miguel A. Valvano, Kun Zhang, Gerard D. Wright, Fiona Mcarthur, Kim M Blakely, Gladys P De Leon, John R Walker, Elena Evdokimova, Murray S. JunopAbstract:The barrier imposed by lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria presents a significant challenge in treatment of these organisms with otherwise effective hydrophobic antibiotics. The absence of L-glycero-D-manno-Heptose in the LPS molecule is associated with a dramatically increased bacterial susceptibility to hydrophobic antibiotics and thus enzymes in the ADP-Heptose biosynthesis pathway are of significant interest. GmhA catalyzes the isomerization of D-sedoheptulose 7-phosphate into D-glycero-D-manno-Heptose 7-phosphate, the first committed step in the formation of ADP-Heptose. Here we report structures of GmhA from Escherichia coli and Pseudomonas aeruginosa in apo, substrate, and product-bound forms, which together suggest that GmhA adopts two distinct conformations during isomerization through reorganization of quaternary structure. Biochemical characterization of GmhA mutants, combined with in vivo analysis of LPS biosynthesis and novobiocin susceptibility, identifies key catalytic residues. We postulate GmhA acts through an enediol-intermediate isomerase mechanism.
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An In Vitro Screen of Bacterial Lipopolysaccharide Biosynthetic Enzymes Identifies an Inhibitor of ADP-Heptose Biosynthesis
Chemistry & biology, 2006Co-Authors: Gladys P De Leon, Miguel A. Valvano, Nadine H. Elowe, Kalinka Koteva, Gerard D. WrightAbstract: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.
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biosynthesis pathway of adp l glycero β d manno Heptose in escherichia coli
Journal of Bacteriology, 2002Co-Authors: Bernd Kneidinger, Alla Zamyatina, Paul Kosma, Michael Graninger, Miguel A. Valvano, Cristina L Marolda, Fiona Mcarthur, Paul MessnerAbstract: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.
Dong Hae Shin - One of the best experts on this subject based on the ideXlab platform.
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inhibition of d glycero β d manno Heptose 1 phosphate adenylyltransferase from burkholderia pseudomallei by epigallocatechin gallate and myricetin
Biochemical Journal, 2021Co-Authors: Suwon Kim, Misun Kim, Heejin Kam, Dong Hae ShinAbstract:Flavonoids play beneficial roles in various human diseases. In this study, a flavonoid library was employed to probe inhibitors of d-glycero-β-d-manno-Heptose-1-phosphate adenylyltransferase from Burkholderia pseudomallei (BpHldC) and two flavonoids, epigallocatechin gallate (EGCG) and myricetin, have been discovered. BpHldC is one of the essential enzymes in the ADP-l-glycero-β-d-manno-Heptose biosynthesis pathway constructing lipopolysaccharide of B. pseudomallei. Enzyme kinetics study showed that two flavonoids work through different mechanisms to block the catalytic activity of BpHldC. Among them, a docking study of EGCG was performed and the binding mode could explain its competitive inhibitory mode for both ATP and βG1P. Analyses with EGCG homologs could reveal the important functional moieties, too. This study is the first example of uncovering the inhibitory activity of flavonoids against the ADP-l-glycero-β-d-manno-Heptose biosynthesis pathway and especially targeting HldC. Since there are no therapeutic agents and vaccines available against melioidosis, EGCG and myricetin can be used as templates to develop antibiotics over B. pseudomallei.
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A Study of a Potent Inhibitor Against a GDP-6-Deoxy-α-d-Manno-Heptose Biosynthesis Pathway as Antibiotic Candidates
Microbial drug resistance (Larchmont N.Y.), 2019Co-Authors: Suwon Kim, Misun Kim, Dong Hae ShinAbstract:The GDP-6-deoxy-α-d-manno-Heptose is a key building block molecule in constructing lipopolysaccharide of Gram-negative bacteria. Therefore, blockage of the biosynthesis pathway of GDP-6-deoxy-α-d-m...
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crystal structure of d glycero β d manno Heptose 1 phosphate adenylyltransferase from burkholderia pseudomallei
Proteins, 2018Co-Authors: Jimin Park, Daeun Lee, Hyojin Kim, Suwon Kim, Misun Kim, Dong Hae ShinAbstract:The crystal structure of HldC from B. pseudomallei (BpHldC), the fourth enzyme of the Heptose biosynthesis pathway, has been determined. BpHldC converts ATP and d-glycero-β-d-manno-Heptose-1-phosphate into ADP-d-glycero-β-d-manno-Heptose and pyrophosphate. The crystal structure of BpHldC belongs to the nucleotidyltransferase α/β phosphodiesterase superfamily sharing a common Rossmann-like α/β fold with a conserved T/HXGH sequence motif. The invariant catalytic key residues of BpHldC indicate that the core catalytic mechanism of BpHldC may be similar to that of other closest homologues. Intriguingly, a reorientation of the C-terminal helix seems to guide open and close states of the active site for the catalytic reaction.
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Crystal structure of d-glycero-α-d-manno-Heptose-1-phosphate guanylyltransferase from Yersinia pseudotuberculosis.
Biochimica et biophysica acta. Proteins and proteomics, 2017Co-Authors: Hyojin Kim, Jimin Park, Suwon Kim, Dong Hae ShinAbstract:Abstract The Gram-negative bacterium Yersinia pseudotuberculosis is the causative agent of yersiniosis. d -glycero-α- d -manno-Heptose-1-phosphate guanylyltransferase (HddC) is the fourth enzyme of the GDP- d -glycero-α- d -manno-Heptose biosynthesis pathway which is important for the virulence of the microorganism. Therefore, HddC is a potential target of antibiotics against yersiniosis. In this study, HddC from the synthesized HddC gene of Y. pseudotuberculosis has been expressed, purified, crystallized. Synchrotron X-ray data from a selenomethionine-substituted HddC crystal were also collected and its structure was determined at 2.0 A resolution. Structure analyses revealed that it belongs to the glycosyltransferase A type superfamily members with the signature motif GXGXR for nucleotide binding. Despite of remarkable structural similarity, HddC uses GTP for catalysis instead of CTP and UTP which are used for other major family members, cytidylyltransferase and uridylyltransferase, respectively. We suggest that EXXPLGTGGA and L(S/A/G)X(S/G) motifs are probably essential to bind with GTP and a FSFE motif with substrate.
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General assay for enzymes in the Heptose biosynthesis pathways using electrospray ionization mass spectrometry
Applied microbiology and biotechnology, 2017Co-Authors: Jimin Park, Daeun Lee, Eun-kyoung Seo, Jae-sang Ryu, Dong Hae ShinAbstract:The ADP-l-glycero-β-d-manno-Heptose and the GDP-6-deoxy-α-d-manno-Heptose biosynthesis pathways play important roles in constructing lipopolysaccharide of Gram-negative bacteria. Blocking the pathways is lethal or increases antibiotic susceptibility to pathogens. Therefore, the enzymes involved in the pathways are novel antibiotic drug targets. Here, we designed an efficient method to assay the whole enzymes in the pathways using mass spectrometry and screened 148 compounds. One promising lead is (−)-nyasol targeting d-glycero-α-d-manno-Heptose-1-phosphate guanylyltransferase (HddC) included in the GDP-6-deoxy-α-d-manno-Heptose biosynthesis pathway from Burkholderia pseudomallei. The inhibitory activity of the lead compound against HddC has been confirmed by blocking the system transferring the guanosine monophosphate (GMP) moiety to α-d-glucose-1-phosphate. (−)-Nyasol exhibits the half maximal inhibitory concentration (IC50) value of 17.6 μM. A further study is going on using (−)-nyasol derivatives to find better leads with high affinity.
