Show legend Show as text

Structure type: monomer
Trivial name: UDP-2-acetamido-4-amino-2,4,6-trideoxy-β-L-altropyranose
Compound class: nucleoside diphosphate sugar

• Article ID: 3184
  Schoenhofen IC, McNally DJ, Vinogradov E, Whitfield D, Young NM, Dick S, Wakarchuk WW, Brisson JR, Logan SM "Functional characterization of dehydratase/aminotransferase pairs from Helicobacter and Campylobacter: enzymes distinguishing the pseudaminic acid and bacillosamine biosynthetic pathways" - Journal of Biological Chemistry 281(2) (2006) 723-732
  

  Helicobacter pylori and Campylobacter jejuni have been shown to modify their flagellins with pseudaminic acid (Pse), via O-linkage, while C. jejuni also possesses a general protein glycosylation pathway (Pgl) responsible for the N-linked modification of at least 30 proteins with a heptasaccharide containing 2,4-diacetamido-2,4,6-trideoxy-α-D-glucopyranose, a derivative of bacillosamine. To further define the Pse and bacillosamine biosynthetic pathways, we have undertaken functional characterization of UDP-α-D-GlcNAc modifying dehydratase/aminotransferase pairs, in particular the H. pylori and C. jejuni flagellar pairs HP0840/HP0366 and Cj1293/Cj1294, as well as the C. jejuni Pgl pair Cj1120c/Cj1121c using His(6)-tagged purified derivatives. The metabolites produced by these enzymes were identified using NMR spectroscopy at 500 and/or 600 MHz with a cryogenically cooled probe for optimal sensitivity. The metabolites of Cj1293 (PseB) and HP0840 (FlaA1) were found to be labile and could only be characterized by NMR analysis directly in aqueous reaction buffer. The Cj1293 and HP0840 enzymes exhibited C6 dehydratase as well as a newly identified C5 epimerase activity that resulted in the production of both UDP-2-acetamido-2,6-dideoxy-β-L-arabino-4-hexulose and UDP-2-acetamido-2,6-dideoxy-α-D-xylo-4-hexulose. In contrast, the Pgl dehydratase Cj1120c (PglF) was found to possess only C6 dehydratase activity generating UDP-2-acetamido-2,6-dideoxy-α-D-xylo-4-hexulose. Substrate-specificity studies demonstrated that the flagellar aminotransferases HP0366 and Cj1294 utilize only UDP-2-acetamido-2,6-dideoxy-β-L-arabino-4-hexulose as substrate producing UDP-4-amino-4,6-dideoxy-β-L-AltNAc, a precursor in the Pse biosynthetic pathway. In contrast, the Pgl aminotransferase Cj1121c (PglE) utilizes only UDP-2-acetamido-2,6-dideoxy-α-D-xylo-4-hexulose producing UDP-4-amino-4,6-dideoxy-α-D-GlcNAc (UDP-2-acetamido-4-amino-2,4,6-trideoxy-α-D-glucopyranose), a precursor used in the production of the Pgl glycan component 2,4-diacetamido-2,4,6-trideoxy-α-D-glucopyranose.

  Campylobacter, NMR spectroscopy, pseudaminic acid, Helicobacter pylori, Substrate Specificity, glycosylation, bacillosamine, Flagellin

  NCBI PubMed ID: 16286454
  Publication DOI: 10.1074/jbc.M511021200
  Journal NLM ID: 2985121R
  Publisher: Baltimore, MD: American Society for Biochemistry and Molecular Biology
  Correspondence: susan.logan@nrc-cnrc.gc.ca
  Institutions: Institute for Biological Sciences, National Research Council, Ottawa, Ontario, Canada
  Methods: NMR-2D, NMR, SDS-PAGE, genetic methods, biochemical methods, capillary electrophoresis (CE)
  
   
  
  Helicobacter pylori HP0366, Campylobacter jejuni Cj1294
  CSDB ID 20536 (all data & tools)
• Article ID: 3196
  Schoenhofen IC, Lunin VV, Julien JP, Li Y, Ajamian E, Matte A, Cygler M, Brisson JR, Aubry A, Logan SM, Bhatia S, Wakarchuk WW, Young NM "Structural and functional characterization of PseC, an aminotransferase involved in the biosynthesis of pseudaminic acid, an essential flagellar modification in Helicobacter pylori" - Journal of Biological Chemistry 281(13) (2006) 8907-8916
  

  Helicobacter pylori flagellin is heavily glycosylated with the novel sialic acid-like nonulosonate, pseudaminic acid (Pse). The glycosylation process is essential for assembly of functional flagellar filaments and consequent bacterial motility. Because motility is a key virulence factor for this and other important pathogens, the Pse biosynthetic pathway offers potential for novel therapeutic targets. From recent NMR analyses, we determined that the conversion of UDP-α-D-Glc-NAc to the central intermediate in the pathway, UDP-4-amino-4,6-dideoxy-β-L-AltNAc, proceeds by formation of UDP-2-acetamido-2,6-dideoxy-β-L-arabino-4-hexulose by the dehydratase/epimerase PseB (HP0840) followed with amino transfer by the aminotransferase, PseC (HP0366). The central role of PseC in the H. pylori Pse biosynthetic pathway prompted us to determine crystal structures of the native protein, its complexes with pyridoxal phosphate alone and in combination with the UDP-4-amino-4,6-dideoxy-β-L-AltNAc product, the latter being converted to the external aldimine form in the active site of the enzyme. In the binding site, the AltNAc sugar ring adopts a (4)C(1) chair conformation, which is different from the predominant (1)C(4) form found in solution. The enzyme forms a homodimer where each monomer contributes to the active site, and these structures have permitted the identification of key residues involved in stabilization, and possibly catalysis, of the β-L-arabino intermediate during the amino transfer reaction. The essential role of Lys(183) in the catalytic event was confirmed by site-directed mutagenesis. This work presents for the first time a nucleotide-sugar aminotransferase co-crystallized with its natural ligand, and, in conjunction with the recent functional characterization of this enzyme, these results will assist in elucidating the aminotransferase reaction mechanism within the Pse biosynthetic pathway.

  biosynthesis, pseudaminic acid, Helicobacter pylori, Flagellin, aminotransferase, PseC

  NCBI PubMed ID: 16421095
  Publication DOI: 10.1074/jbc.M512987200
  Journal NLM ID: 2985121R
  Publisher: Baltimore, MD: American Society for Biochemistry and Molecular Biology
  Correspondence: Martin.Young@nrc-cnrc.gc.ca
  Institutions: Institute for Biological Sciences, National Research Council, Ottawa, Ontario K1A 0R6, Biotechnology Research, Institute, National Research Council, Montreal, Quebec H4P 2R2, Department of Biochemistry, McGill University, Montreal, Quebec H3G 1Y6, Canada
  Methods: NMR-2D, NMR, SDS-PAGE, genetic methods, biochemical methods, capillary electrophoresis (CE), crystallization
  
   
  
  Helicobacter pylori 26695/1061
  CSDB ID 20543 (all data & tools)
• Article ID: 3404
  Guerry P, Ewing CP, Schoenhofen IC, Logan SM "Protein glycosylation in Campylobacter jejuni: partial suppression of pglF by mutation of pseC" - Journal of Bacteriology 189(18) (2007) 6731-6733
  

  Campylobacter jejuni has systems for N- and O-linked protein glycosylation. Although biochemical evidence demonstrated that a pseC mutant in the O-linked pathway accumulated the product of pglF in the N-linked pathway, analyses of transformation frequencies and glycosylation statuses of N-glycosylated proteins indicated a partial suppression of pglF by pseC

  biosynthesis, Bacterial Proteins, Campylobacter jejuni, glycosylation

  NCBI PubMed ID: 17631632
  Journal NLM ID: 2985120R
  Publisher: American Society for Microbiology
  Correspondence: guerryp@nmrc.navy.mil
  Institutions: Enteric Diseases Department, Naval Medical Research Center, 503 Robert Grant Ave., Silver Spring, MD 20910, USA
  Methods: PCR, serological methods, genetic methods
  
   
  
  Campylobacter jejuni
  CSDB ID 21964 (all data & tools)
• Article ID: 3506
  Liu B, Knirel YA, Feng L, Perepelov AV, Senchenkova SN, Wang Q, Reeves P, Wang L "Structure and genetics of Shigella O antigens" - FEMS Microbiology Reviews 32(4) (2008) 627-653
  

  This review covers the O antigens of the 46 serotypes of Shigella, but those of most Shigella flexneri are variants of one basic structure, leaving 34 Shigella distinct O antigens to review, together with their gene clusters. Several of the structures and gene clusters are reported for the first time and this is the first such group for which structures and DNA sequences have been determined for all O antigens. Shigella strains are in effect Escherichia coli with a specific mode of pathogenicity, and 18 of the 34 O antigens are also found in traditional E. coli. Three are very similar to E. coli O antigens and 13 are unique to Shigella strains. The O antigen of Shigella sonnei is quite atypical for E. coli and is thought to have transferred from Plesiomonas. The other 12 O antigens unique to Shigella strains have structures that are typical of E. coli, but there are considerably more anomalies in their gene clusters, probably reflecting recent modification of the structures. Having the complete set of structures and genes opens the way for experimental studies on the role of this diversity in pathogenicity.

  structure, O antigen, Shigella, O antigen gene cluster, O antigen diversity

  NCBI PubMed ID: 18422615
  Publication DOI: 10.1111/j.1574-6976.2008.00114.x
  Journal NLM ID: 8902526
  Publisher: Oxford University Press
  Correspondence: wanglei@nankai.edu.cn
  Institutions: TEDA School of Biological Sciences and Biotechnology, Nankai University, TEDA, Tianjin, China, TEDA School of Biological Sciences and Biotechnology, Nankai University, TEDA, Tianjin, China.
  Methods: 13C NMR, 1H NMR, NMR-2D, sugar analysis, ESI-MS, serological methods, genetic methods, biochemical methods
  
   
  
  Shigella
  CSDB ID 23111 (all data & tools)
• Article ID: 3767
  Rangarajan ES, Proteau A, Cui Q, Logan SM, Potetinova Z, Whitfield D, Purisima EO, Cygler M, Matte A, Sulea T, Schoenhofen IC "Structural and Functional Analysis of Campylobacter jejuni PseG: A UDP-sugar hydrolase from the pseudaminic acid biosynthetic pathway" - Journal of Biological Chemistry 284(31) (2009) 20989-21000
  

  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 A resolution, respectively. In addition, molecular modeling was utilized to provide insight into the structure of the PseG-substrate complex. This modeling identifies a His(17)-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 His(17) 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.

  Campylobacter jejuni, pseudaminic acid, crystal structure, Helicobacter pylori, glycosyltransferase, modeling, hydrolase, flagella

  NCBI PubMed ID: 19483088
  Journal NLM ID: 2985121R
  Publisher: Baltimore, MD: American Society for Biochemistry and Molecular Biology
  Correspondence: traian.sulea@nrc-cnrc.gc.ca; ian.schoenhofen@nrc-cnrc.gc.ca
  Institutions: From the Department of Biochemistry, McGill University, Montreal, Quebec H3G 1V6
  Methods: X-ray, SDS-PAGE, MD simulations, genetic methods, molecular modeling, CD, crystallization
  
   
  
  Campylobacter jejuni 11168
  CSDB ID 24190 (all data & tools)
• Article ID: 4582
  Mostafavi AZ, Troutman JM "Biosynthetic assembly of the Bacteroides fragilis capsular polysaccharide A precursor bactoprenyl diphosphate-linked acetamido-4-amino-6-deoxygalactopyranose" - Biochemistry 52(11) (2013) 1939-1949
  

  The sugar capsule capsular polysaccharide A (CPSA), which coats the surface of the mammalian symbiont Bacteroides fragilis, is a key mediator of mammalian immune system development. In addition, this sugar polymer has shown therapeutic potential in animal models of multiple sclerosis and other autoimmune disorders. The structure of the CPSA polymer includes a rare stereoconfiguration sugar acetamido-4-amino-6-deoxygalactopyranose (AADGal) that we propose is the first sugar linked to a bactoprenyl diphosphate scaffold in the production of CPSA. In this report, we have utilized a heterologous system to reconstitute bactoprenyl diphosphate-linked AADGal production. Construction of this system included a previously reported Campylobacter jejuni dehydratase, PglF, coupled to a B. fragilis-encoded aminotransferase (WcfR) and initiating hexose-1-phosphate transferase (WcfS). The function of the aminotransferase was confirmed by capillary electrophoresis and a novel high-performance liquid chromatography (HPLC) method. Production of the rare uridine diphosphate (UDP)-AADGal was confirmed through a series of one- and two-dimensional nuclear magnetic resonance experiments and high-resolution mass spectrometry. A spectroscopically unique analogue of bactoprenyl phosphate was utilized to characterize the transfer reaction catalyzed by WcfS and allowed HPLC-based isolation of the isoprenoid-linked sugar product. Importantly, the entire heterologous system was utilized in a single-pot reaction to biosynthesize the bactoprenyl-linked sugar. This work provides the first critical step in the in vitro reconstitution of CPSA biosynthesis.

  Bacteroides fragilis, aminotransferase, caspsular polysaccharide A, biosynthesis pathway, WcfR, WcfS, acetamido-4-amino-6-deoxy-galactopyranose

  NCBI PubMed ID: 23458065
  Publication DOI: 10.1021/bi400126w
  Journal NLM ID: 0370623
  Publisher: American Chemical Society
  Correspondence: jerry.troutman@uncc.edu
  Institutions: Department of Chemistry, University of North Carolina at Charlotte , 9201 University City Boulevard, Charlotte, North Carolina 28223-0001, United States
  Methods: 1H NMR, NMR-2D, PCR, SDS-PAGE, TLC, ESI-MS, NMR-1D, genetic methods, biochemical methods, radioactivity measurement, HPLC, CE
  
   
  
  Campylobacter jejuni
  CSDB ID 29658 (all data & tools)

Show legend Show as text

Structure type: monomer
Trivial name: UDP-2,4-diacetamido-2,4,6-trideoxy-β-L-altropyranose

• Article ID: 3506
  Liu B, Knirel YA, Feng L, Perepelov AV, Senchenkova SN, Wang Q, Reeves P, Wang L "Structure and genetics of Shigella O antigens" - FEMS Microbiology Reviews 32(4) (2008) 627-653
  

  This review covers the O antigens of the 46 serotypes of Shigella, but those of most Shigella flexneri are variants of one basic structure, leaving 34 Shigella distinct O antigens to review, together with their gene clusters. Several of the structures and gene clusters are reported for the first time and this is the first such group for which structures and DNA sequences have been determined for all O antigens. Shigella strains are in effect Escherichia coli with a specific mode of pathogenicity, and 18 of the 34 O antigens are also found in traditional E. coli. Three are very similar to E. coli O antigens and 13 are unique to Shigella strains. The O antigen of Shigella sonnei is quite atypical for E. coli and is thought to have transferred from Plesiomonas. The other 12 O antigens unique to Shigella strains have structures that are typical of E. coli, but there are considerably more anomalies in their gene clusters, probably reflecting recent modification of the structures. Having the complete set of structures and genes opens the way for experimental studies on the role of this diversity in pathogenicity.

  structure, O antigen, Shigella, O antigen gene cluster, O antigen diversity

  NCBI PubMed ID: 18422615
  Publication DOI: 10.1111/j.1574-6976.2008.00114.x
  Journal NLM ID: 8902526
  Publisher: Oxford University Press
  Correspondence: wanglei@nankai.edu.cn
  Institutions: TEDA School of Biological Sciences and Biotechnology, Nankai University, TEDA, Tianjin, China, TEDA School of Biological Sciences and Biotechnology, Nankai University, TEDA, Tianjin, China.
  Methods: 13C NMR, 1H NMR, NMR-2D, sugar analysis, ESI-MS, serological methods, genetic methods, biochemical methods
  
   
  
  Shigella
  CSDB ID 23112 (all data & tools)
• Article ID: 3767
  Rangarajan ES, Proteau A, Cui Q, Logan SM, Potetinova Z, Whitfield D, Purisima EO, Cygler M, Matte A, Sulea T, Schoenhofen IC "Structural and Functional Analysis of Campylobacter jejuni PseG: A UDP-sugar hydrolase from the pseudaminic acid biosynthetic pathway" - Journal of Biological Chemistry 284(31) (2009) 20989-21000
  

  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 A resolution, respectively. In addition, molecular modeling was utilized to provide insight into the structure of the PseG-substrate complex. This modeling identifies a His(17)-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 His(17) 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.

  Campylobacter jejuni, pseudaminic acid, crystal structure, Helicobacter pylori, glycosyltransferase, modeling, hydrolase, flagella

  NCBI PubMed ID: 19483088
  Journal NLM ID: 2985121R
  Publisher: Baltimore, MD: American Society for Biochemistry and Molecular Biology
  Correspondence: traian.sulea@nrc-cnrc.gc.ca; ian.schoenhofen@nrc-cnrc.gc.ca
  Institutions: From the Department of Biochemistry, McGill University, Montreal, Quebec H3G 1V6
  Methods: X-ray, SDS-PAGE, MD simulations, genetic methods, molecular modeling, CD, crystallization
  
   
  
  Campylobacter jejuni 11168
  CSDB ID 24191 (all data & tools)

Show legend Show as text

Structure type: monomer
Trivial name: UDP-4-amino-6-deoxy-L-AltNAc (UDP-4-amino-4,6-dideoxy-N-acetyl-β-L-altrosamine)
Contained glycoepitopes: IEDB_141494

• Article ID: 4770
  Li Z, Hwang S, Ericson J, Bowler K, Bar-Peled M "Pen and Pal Are Nucleotide-Sugar Dehydratases That Convert UDP-GlcNAc to UDP-6-Deoxy-D-GlcNAc-5,6-ene and Then to UDP-4-Keto-6-deoxy-L-AltNAc for CMP-Pseudaminic Acid Synthesis in Bacillus thuringiensis" - Journal of Biological Chemistry 290(2) (2015) 691-704
  

  CMP-pseudaminic acid is a precursor required for the O-glycosylation of flagellin in some pathogenic Gram-negative bacteria, a process known to be critical in bacterial motility and infection. However, little is known about flagellin glycosylation in Gram-positive bacteria. Here, we identified and functionally characterized an operon, named Bti_pse, in Bacillus thuringiensis israelensis ATCC 35646, which encodes seven different enzymes that together convert UDP-GlcNAc to CMP-pseudaminic acid. In contrast, Gram-negative bacteria complete this reaction with six enzymes. The first enzyme, which we named Pen, converts UDP-D-GlcNAc to an uncommon UDP-sugar, UDP-6-deoxy-D-GlcNAc-5,6-ene. Pen contains strongly bound NADP+ and has distinct UDP-GlcNAc 4-oxidase, 5,6-dehydratase, and 4-reductase activities. The second enzyme, which we named Pal, converts UDP-6-deoxy-D-GlcNAc-5,6-ene to UDP-4-keto-6-deoxy-L-AltNAc. Pal is NAD+-dependent and has distinct UDP-6-deoxy-D-GlcNAc-5,6-ene 4-oxidase, 5,6-reductase, and 5-epimerase activities. We also show here using NMR spectroscopy and mass spectrometry that in B. thuringiensis, the enzymatic product of Pen and Pal, UDP-4-keto-6-deoxy-L-AltNAc, is converted to CMP-pseudaminic acid by the sequential activities of a C4?-transaminase (Pam), a 4-N-acetyltransferase (Pdi), a UDP-hydrolase (Phy), an enzyme (Ppa) that adds phosphoenolpyruvate to form pseudaminic acid, and finally a cytidylyltransferase that condenses CTP to generate CMP-pseudaminic acid. Knowledge of the distinct dehydratase-like enzymes Pen and Pal and their role in CMP-pseudaminic acid biosynthesis in Gram-positive bacteria provides a foundation to investigate the role of pseudaminic acid and flagellin glycosylation in Bacillus and their involvement in bacterial motility and pathogenicity.

  pseudaminic acid, Bacillus, aminotransferase, CMP-pseudaminic acid, flagellin glycosylation, dehydratase, enzyme catalysis, bacterial metabolism, Bacillus thuringiensis, carbohydrate biosynthesis, glycobiology, UDP-GlcNAc

  NCBI PubMed ID: 25414257
  Publication DOI: 10.1074/jbc.M114.612747
  Journal NLM ID: 2985121R
  Publisher: Baltimore, MD: American Society for Biochemistry and Molecular Biology
  Correspondence: peled@ccrc.uga.edu
  Institutions: Complex Carbohydrate Research Center, University of Georgia, Athens, GA, USA, From the Department of Plant Biology and Complex Carbohydrate Research Center, University of Georgia, Athens, GA, USA
  Methods: 13C NMR, 1H NMR, gel filtration, NMR-2D, PCR, SDS-PAGE, DNA techniques, kinetics assays, NMR-1D, genetic methods, biochemical methods, HPLC, UV, enzymatic analysis, LC-ESI-MS/MS, HILIC-HPLC-UV, HILIC-ESI-MS
  
   
  
  Bacillus thuringiensis sv. israelensis ATCC 35646
  CSDB ID 30805 (all data & tools)