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Structure type: monomer
Trivial name: dTDP-6-deoxy-D-xylohex-4-ulose, dTDP-4-keto-6-deoxy-α-D-glucose, dTDP-6-dideoxy-xylo-hexos-4-ulose, dTDP-6-deoxy-α-D-xylo-hex-4-ulose, dTDP-4-oxo-6-deoxy-α-D-glucose, dTDP-6-deoxy-D-xylo-hex-4-ulopyranose, dTDP-4-keto-6-deoxy-D-glucose, dTDP-4-keto-6-deoxyglucose
Compound class: nucleoside diphosphate sugar
Contained glycoepitopes: IEDB_138113,IEDB_196259

• Article ID: 349
  Pfoestl A, Hofinger A, Kosma P, Messner P "Biosynthesis of dTDP-3-acetamido-3,6-dideoxy-a-D-galactose in Aneurinibacillus thermoaerophilus L420-91T" - Journal of Biological Chemistry 278(29) (2003) 26410-26417
  

  The glycan chain of the S-layer protein of Aneurinibacillus thermoaerophilus L420-91T (DSM 10154) consists of d-rhamnose and 3-acetamido-3,6-dideoxy-d-galactose (d-Fucp3NAc). Thymidine diphosphate-activated d-Fucp3NAc serves as precursor for the assembly of structural polysaccharides in Gram-positive and Gram-negative organisms. The biosynthesis of dTDP-3-acetamido-3,6-dideoxy-α-D-galactose (dTDP-d-Fucp3NAc) involves five enzymes. The first two steps of the reaction are catalyzed by enzymes that are part of the well studied dTDP-l-rhamnose biosynthetic pathway, namely d-glucose-1-phosphate thymidyltransferase (RmlA) and dTDP-d-glucose-4,6-dehydratase (RmlB). The enzymes catalyzing the last three synthesis reactions have not been characterized biochemically so far. These steps include an isomerase, a transaminase, and a transacetylase. We identified all five genes involved by chromosome walking in the Gram-positive organism A. thermoaerophilus L420-91T and overexpressed the three new enzymes heterologously in Escherichia coli. The activities of these enzymes were monitored by reverse phase high performance liquid chromatography, and the intermediate products formed were characterized by 1H and 13C nuclear magnetic resonance spectroscopy analysis. Alignment of the newly identified proteins with known sequences revealed that the elucidated pathway in this Gram-positive organism may also be valid in the biosynthesis of the O-antigen of lipopolysaccharides of Gram-negative organisms. The key enzyme in the biosynthesis of dTDP-d-Fucp3NAc has been identified as an isomerase, which converts the 4-keto educt into the 3-keto product, with concomitant epimerization at C-4 to produce a 6-deoxy-d-xylo configuration. This is the first report of the functional characterization of the biosynthesis of dTDP-d-Fucp3NAc and description of a novel type of isomerase capable of synthesizing dTDP-6-deoxy-d-xylohex-3-ulose from dTDP-6-deoxy-d-xylohex-4-ulose.

  biosynthesis, 6-dideoxy-D-galactose, 3-Acetamido-3, Aneurinibacillus, Fuc3NAc

  NCBI PubMed ID: 12740380
  Journal NLM ID: 2985121R
  Publisher: Baltimore, MD: American Society for Biochemistry and Molecular Biology
  Correspondence: paul.messner@boku.ac.at
  Institutions: Zentrum für Ultrastrukturforschung und Ludwig Boltzmann-Institut fur Molekulare Nanotechnologie, Universitat fur Bodenkultur Wien, A-1180 Wien, Austria, Institut fur Chemie, Universitat fur Bodenkultur Wien, A-1190 Wien, Austria
  Methods: NMR-2D, NMR, biosynthetic methods
  
   
  
  Aneurinibacillus thermoaerophilus L420-91T
  CSDB ID 348 (all data & tools)
• Article ID: 3181
  Kang YB, Yang YH, Lee KW, Lee SG, Sohng JK, Lee HC, Liou K, Kim BG "Preparative synthesis of dTDP-L-rhamnose through combined enzymatic pathways" - Biotechnology and Bioengineering 93(1) (2006) 21-27
  

  dTDP-L-rhamnose, an important precursor of O-antigen, was prepared on a large scale from dTMP by executing an one-pot reaction in which six enzymes are involved. Two enzymes, dTDP-4-keto-6-deoxy-D-glucose 3,5-epimerase and dTDP-4-keto-rhamnose reductase, responsible for the conversion of dTDP-4-keto-6-deoxy-D-glucose to dTDP-L-rhamnose, were isolated from their putative sequences in the genome of Mesorhizobium loti, functionally expressed in Escherichia coli, and their enzymatic activities were identified. The two enzymes were combined with an enzymatic process for dTDP-4-keto-6-deoxy-D-glucose involving TMP kinase, acetate kinase, dTDP-glucose synthase, and dTDP-glucose 4,6-dehydratase, which allowed us to achieve a preparative scale synthesis of dTDP-L-rhamnose using dTMP and glucose-1-phosphate as starting materials. About 82% yield of dTDP-L-rhamnose was obtained based on initial dTMP concentration at 20 mM dTMP, 1 mM ATP, 10 mM NADH, 60 mM acetyl phosphate, and 80 mM glucose-1-phosphate. From the reaction with 20 ml volume, approximately 180 mg of dTDP-L-rhamnose was obtained in an overall yield of 60% after two-step purification, that is, anion exchange chromatography and gel filtration for desalting. The purified product was identified by HPLC, ESI-MS, and NMR, showing about 95% purity.

  dTDP-L-rhamnose, dTDP-4-keto-6-deoxy-D-glucose, dTDP-4-keto-6-deoxy-D-glucose 3, 5-epimerase, dTDP-4-keto-rhamnose reductase

  NCBI PubMed ID: 16276532
  Publication DOI: 10.1002/bit.20648
  Journal NLM ID: 7502021
  Publisher: New York: Wiley-VCH
  Correspondence: byungkim@snu.ac.kr
  Institutions: School of Chemical and Biological Engineering, Seoul National University, Korea, Interdisciplinary Program for Biochemical Engineering and Biotechnology, Seoul National University, Seoul, 151-742, Korea, Department of Chemical and Biochemical Engineering, Pusan National University, Busan, 609-735, Korea, Institute of Biomolecule Reconstruction, Sunmoon University, Asan, 336-840, Korea
  Methods: 13C NMR, 1H NMR, ESI-MS, NMR-1D, genetic methods
  
   
  
  Escherichia coli MC1061, Salmonella enterica typhimurium LT2
  CSDB ID 20526 (all data & tools)
• Article ID: 3322
  Wang Y, Xu Y, Perepelov AV, Qi Y, Knirel YA, Wang L, Feng L "Biochemical characterization of dTDP-D-Qui4N and dTDP-D-Qui4NAc biosynthetic pathways in Shigella dysenteriae type 7 and Escherichia coli O7" - Journal of Bacteriology 189(23) (2007) 8626-8635
  

  O antigen variation due to the presence of different types of sugars and sugar linkages is important for the survival of bacteria against host immune systems. The O antigens of S. dysenteriae type 7 and E. coli O7 contain 4-(N-acetylglycyl)amino-4,6-dideoxy-D-glucose (D-Qui4NGlyAc) and 4-acetamido-4,6-dideoxy-D-glucose (D-Qui4NAc), respectively, which are sugars not often found in studied polysaccharides. In this study, we characterized the biosynthetic pathways for dTDP-D-Qui4N and dTDP-D-Qui4NAc (the nucleotide-activated precursors of D-Qui4NGlyAc and D-Qui4NAc in O antigens). Predicted genes involved in the synthesis of the two sugars were cloned, and the gene products were overexpressed and purified as His-tagged fusion proteins. In vitro enzymatic reactions were carried out using the purified proteins, and the reaction products were analyzed by capillary electrophoresis, electrospray ionization mass spectrometry, and nuclear magnetic resonance spectroscopy. It was shown that in S. dysenteriae type 7 and E. coli O7 dTDP-D-Qui4N is synthesized from α-D-glucose-1-phosphate in three reaction steps catalyzed by glucose-1-phosphate thymidyltransferase (RmlA), dTDP-D-glucose 4,6-dehydratase (RmlB), and dTDP-4-keto-6-deoxy-D-glucose aminotransferase (VioA), respectively. An additional acetyltransferase (VioB) catalyzes the conversion of dTDP-D-Qui4N into dTDP-D-Qui4NAc in E. coli O7. Kinetic parameters and some other properties of VioA and VioB are described and differences between VioA proteins from S. dysenteriae type 7 and E. coli O7 discussed. To our knowledge, this is the first time that functions of VioA and VioB have been biochemically characterized. This study provides valuable enzyme sources for the production of dTDP-D-Qui4N and dTDP-D-Qui4NAc, which are potentially useful in pharmaceutical industry for drugs development

  biosynthesis, Escherichia coli, Shigella dysenteriaes

  NCBI PubMed ID: 17905981
  Journal NLM ID: 2985120R
  Publisher: American Society for Microbiology
  Correspondence: fenglu63@nankai.edu.cn
  Institutions: TEDA School of Biological Sciences and Biotechnology, Nankai University,1 Tianjin Key Laboratory of Microbial Functional Genomics, Tianjin Research Center for Functional Genomics and Biochip, 23 Hongda Street, TEDA, Tianjin 300457, People’s Republic ofChina, N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia, TEDA School of Biological Sciences and Biotechnology, Nankai University,1 Tianjin Key Laboratory of Microbial Functional Genomics, Tianjin Research Center for Functional Genomics and Biochip, 23 Hongda Street, TEDA, Tianjin 300457, People’s Republic of China, N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia
  Methods: 1H NMR, NMR-2D, 31P NMR, ESI-MS, MS, genetic methods, biochemical methods, HPLC, capillary electrophoresis (CE)
  
   
  
  Shigella dysenteriae 7, Escherichia coli O7
  CSDB ID 21690 (all data & tools)
• Article ID: 3413
  Dong C, Major LL, Srikannathasan V, Errey JC, Giraud MF, Lam JS, Graninger M, Messner P, McNeil MR, Field RA, Whitfield C, Naismith JH "RmlC, a C3' and C5' carbohydrate epimerase, appears to operate via an intermediate with an unusual twist boat conformation" - Journal of Molecular Biology 365(1) (2007) 146-159
  

  The striking feature of carbohydrates is their constitutional, conformational and configurational diversity. Biology has harnessed this diversity and manipulates carbohydrate residues in a variety of ways, one of which is epimerization. RmlC catalyzes the epimerization of the C3' and C5' positions of dTDP-6-deoxy-D-xylo-4-hexulose, forming dTDP-6-deoxy-L-lyxo-4-hexulose. RmlC is the third enzyme of the rhamnose pathway, and represents a validated anti-bacterial drug target. Although several structures of the enzyme have been reported, the mechanism and the nature of the intermediates have remained obscure. Despite its relatively small size (22 kDa), RmlC catalyzes four stereospecific proton transfers and the substrate undergoes a major conformational change during the course of the transformation. Here we report the structure of RmlC from several organisms in complex with product and product mimics. We have probed site-directed mutants by assay and by deuterium exchange. The combination of structural and biochemical data has allowed us to assign key residues and identify the conformation of the carbohydrate during turnover. Clear knowledge of the chemical structure of RmlC reaction intermediates may offer new opportunities for rational drug design

  X-ray crystallography, Drug design, site directed mutagenesis, epimerization; enzyme

  NCBI PubMed ID: 17046787
  Journal NLM ID: 2985088R
  Publisher: Elsevier
  Correspondence: naismith@stand.ac.uk
  Institutions: Centre for Biomolecular Sciences, The University, St. Andrews KY16 9ST, UK
  Methods: biochemical methods, crystallization, ITC
  
   
  
  Mycobacterium tuberculosis, Pseudomonas aeruginosa
  CSDB ID 21584 (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 23100 (all data & tools)
• Article ID: 3536
  Pfostl A, Zayni S, Hofinger A, Kosma P, Schäffer C, Messner P "Biosynthesis of dTDP-3-acetamido-3,6-dideoxy-a-D-glucose" - Biochemical Journal 410(1) (2008) 187-194
  

  Derivatives of 3-amino-3,6-dideoxyhexoses are widespread in Nature. They are part of the repeating units of lipopolysaccharide O-antigens, of the glycan moiety of S-layer (bacterial cell surface layer) glycoproteins and also of many antibiotics. In the present study, we focused on the elucidation of the biosynthesis pathway of dTDP-α-D-Quip3NAc (dTDP-3-acetamido-3,6-dideoxy-α-D-glucose) from the Gram-positive, anaerobic, thermophilic organism Thermoanaerobacterium thermosaccharolyticum E207-71, which carries Quip3NAc in its S-layer glycan. The biosynthesis of dTDP-α-D-Quip3NAc involves five enzymes, namely a transferase, a dehydratase, an isomerase, a transaminase and a transacetylase, and follows a pathway similar to that of dTDP-α-D-Fucp3NAc (dTDP-3-acetamido-3,6-dideoxy-α-D-galactose) biosynthesis in Aneurinibacillus thermoaerophilus L420-91(T). The ORFs (open reading frames) of interest were cloned, overexpressed in Escherichia coli and purified. To elucidate the enzymatic cascade, the different products were purified by HPLC and characterized by NMR spectroscopy. The initiating reactions catalysed by the glucose-1-phosphate thymidylyltransferase RmlA and the dTDP-D-glucose-4,6-dehydratase RmlB are well established. The subsequent isomerase was shown to be capable of forming a dTDP-3-oxo-6-deoxy-D-glucose intermediate from the RmlB product dTDP-4-oxo-6-deoxy-D-glucose, whereas the isomerase involved in the dTDP-α-D-Fucp3NAc pathway synthesizes dTDP-3-oxo-6-deoxy-D-galactose. The subsequent reaction steps of either pathway involve a transaminase and a transacetylase, leading to the specific production of nucleotide-activated 3-acetamido-3,6-dideoxy-α-D-glucose and 3-acetamido-3,6-dideoxy-α-D-galactose respectively. Sequence comparison of the ORFs responsible for the biosynthesis of dTDP-α-D-Quip3NAc revealed homologues in Gram-negative as well as in antibiotic-producing Gram-positive bacteria. There is strong evidence that the elucidated biosynthesis pathway may also be valid for LPS (lipopolysaccharide) O-antigen structures and antibiotic precursors

  3, 6-dideoxyhexose, 3-amino-3, bacterial cell surface layer (S-layer) glycoprotein, dTDP-3-acetamido-3, 6-dideoxy-a-D-glucose (dTDP-a-D-Quip3NAc), 4-oxoisomerase, nucleotide-activated sugar precursor, Thermoanaerobacterium thermosaccharolyticum

  NCBI PubMed ID: 17941826
  Publication DOI: 10.1042/BJ20071044
  Journal NLM ID: 2984726R
  Publisher: London, UK : Published by Portland Press on behalf of the Biochemical Society
  Correspondence: paul.messner@boku.ac.at; christina.schaeffer@boku.ac.at
  Institutions: Zentrum fur NanoBiotechnologie, Universitat fur Bodenkultur Wien, Vienna, Austria
  Methods: 13C NMR, 1H NMR, NMR-1D, genetic methods, biochemical methods, RP-HPLC
  
   
  
  Thermoanaerobacterium thermosaccharolyticum E207-71
  CSDB ID 23229 (all data & tools)
• Article ID: 3567
  Wang Q, Ding P, Perepelov AV, Xu Y, Wang Y, Knirel YA, Wang L, Feng L "Characterization of the dTDP-D-fucofuranose biosynthetic pathway in Escherichia coli O52" - Molecular Microbiology 70(6) (2008) 1358-1367
  

  D-Fucofuranose (D-Fucf) is a component of Escherichia coli O52 O antigen. This uncommon sugar is also the sugar moiety of the anticancer drug - gilvocarcin V produced by many streptomycetes. In E. coli O52, rmlA, rmlB, fcf1 and fcf2 were proposed in a previous study by our group to encode the enzymes of the dTDP-d-Fucf (the nucleotide-activated form of d-Fucf) biosynthetic pathway. In this study, Fcf1 and Fcf2 from E. coli O52 were expressed, purified and assayed for their respective activities. Novel product peaks from enzyme-substrate reactions were detected by capillary electrophoresis and the structures of the product compounds were elucidated by electro-spray ionization mass spectrometry and nuclear magnetic resonance spectroscopy. Fcf1 was confirmed to be a dTDP-6-deoxy-d-xylo-hex-4-ulopyranose reductase for the conversion of dTDP-6-deoxy-d-xylo-hex-4-ulopyranose to dTDP-d-fucopyranose (dTDP-D-Fucp), and Fcf2 a dTDP-d-Fucp mutase for the conversion of dTDP-d-Fucp to dTDP-d-Fucf. The Km of Fcf1 for dTDP-6-deoxy-d-xylo-hex-4-ulopyranose was determined to be 0.38 mM, and of Fcf2 for dTDP-d-Fucp to be 3.43 mM. The functional role of fcf1 and fcf2 in the biosynthesis of E. coli O52 O antigen were confirmed by mutation and complementation tests. This is the first time that the biosynthetic pathway of dTDP-d-Fucf has been fully characterized.

  biosynthesis, O antigen, Biosynthetic Pathways, Escherichia coli O52, D-Fucofuranose (D-Fucf)

  NCBI PubMed ID: 19019146
  Journal NLM ID: 8712028
  Publisher: Blackwell Publishing
  Correspondence: fenglu63@nankai.edu.cn
  Institutions: TEDA School of Biological Sciences and Biotechnology, Nankai University, Tianjin, PR China
  Methods: 13C NMR, 1H NMR, NMR-2D, SDS-PAGE, sugar analysis, ESI-MS, GLC, genetic methods, HPLC, capillary electrophoresis (CE)
  
   
  
  Escherichia coli O52
  CSDB ID 23326 (all data & tools)
• Article ID: 3745
  Nguyen LC, Yamamoto M, Ohnishi-Kameyama M, Andi S, Taguchi F, Iwaki M, Yoshida M, Ishii T, Konishi T, Tsunemi K, Ichinose Y "Genetic analysis of genes involved in synthesis of modified 4-amino-4,6-dideoxyglucose in flagellin of Pseudomonas syringae pv. tabaci" - Molecular Genetics and Genomics 282(6) (2009) 595-605
  

  Glycosylation of flagellin contributes to swimming and swarming motilities, adhesion ability, and consequently virulence in Pseudomonas syringae pv. tabaci 6605. Glycans attached to six serine residues are located in the central region of the flagellin polypeptide. The glycan structure at position Ser 201 was recently revealed to consist of two L: -rhamnoses and one modified 4-amino-4,6-dideoxyglucose (viosamine). To clarify the mechanisms for glycosylation of modified viosamine, genes encoding dTDP-viosamine aminotransferase (vioA), dTDP-viosamine acetyltransferase (vioB), and viosamine-derivative transferase (vioT) were isolated and defective mutants were generated. MALDI-TOF-MS analysis of a lysyl endopeptidase-digested peptide including all six glycosylation sites from each flagellin indicated that the molecular masses of the three flagellin mutants were reduced with highly heterogeneous patterns at regular intervals of 146 Da in the mass range from m/z 13,819 to 15,732. The data indicated that the glycopeptides obtained from mutants had glycans consisting only of deoxyhexose instead of the flagellin glycans including the viosamine derivatives determined previously. The motility and virulence on host tobacco leaves were strongly impaired in the ∆vioA mutant and were weakly reduced in the ∆vioB and ∆vioT mutant strains. These results suggest that the genes vioA, vioB, and vioT are essential for glycosylation of flagellin, and accordingly are required for bacterial virulence.

  gene, Pseudomonas syringae, glycopeptide, glycosylation, Flagellin, viosamine

  NCBI PubMed ID: 19787374
  Journal NLM ID: 101093320
  Publisher: Springer
  Correspondence: yuki@cc.okayama-u.ac.jp
  Institutions: The Graduate School of Natural Science and Technology, Okayama University, Tsushima-naka 1-1-1, Kita-ku, Okayama, 700-8530, Japan
  Methods: GC-MS, GC-EI-MS, SDS-PAGE, sugar analysis, DNA techniques, MALDI-TOF MS, genetic methods
  
   
  
  Pseudomonas syringae pv. tabaci
  CSDB ID 24136 (all data & tools)
• Article ID: 4436
  Maki M, Renkonen R "Biosynthesis of 6-deoxyhexose glycans in bacteria" - Glycobiology 14(3) (2004) 1R-15R
  

  After the breakthroughs in genomic sequencing, one of the next challenges remains to understand the molecular biology of other classes of biomolecules, such as protein and lipids, many of which carry specific glycomodification when mediating their biological functions. This review focuses on the 6-deoxyhexose biosynthesis of cell surface glycans of three Gram-negative pathogens, Helicobacter pylori, Pseudomonas aeruginosa, and Actinobacillus actinomycetemcomitans serotype a. 6-Deoxysugars are important functional components of cell surface glycans, and their biosynthetic pathways might be suitable targets for novel interventions of antibacterial chemotherapy.

  rhamnose, fucose, nucleotide sugars, 6-deoxy-talose

  NCBI PubMed ID: 14693916
  Journal NLM ID: 9104124
  Publisher: IRL Press at Oxford University Press
  Correspondence: risto.renkonen@helsinki.fi
  Institutions: Rational Drug Design Program and Department of Bacteriology and Immunology, Biomedicum and Haartman Institute, PO Box 63, FIN-00014 University of Helsinki, Helsinki, Finland
  
   
  
  Helicobacter pylori, Pseudomonas aeruginosa, Actinobacillus actinomycetemcomitans a
  CSDB ID 28845 (all data & tools)
• Article ID: 5022
  Li ZZ, Riegert AS, Goneau MF, Cunningham AM, Vinogradov E, Li J, Schoenhofen IC, Thoden JB, Holden HM, Gilbert M "Characterization of the dTDP-Fuc3N and dTDP-Qui3N biosynthetic pathways in Campylobacter jejuni 81116" - Glycobiology 27(4) (2017) 358-369
  

  The Gram-negative bacterium Campylobacter jejuni 81116 (Penner serotype HS:6) has a class E lipooligosaccharide (LOS) biosynthesis locus containing 19 genes, which encode for 11 putative glycosyltransferases, 1 lipid A acyltransferase and 7 enzymes thought to be involved in the biosynthesis of dideoxyhexosamine (ddHexN) moieties. Although the LOS outer core structure of C. jejuni 81116 is still unknown, recent mass spectrometry analyses suggest that it contains acetylated forms of two ddHexN residues. For this investigation, five of the genes encoding enzymes reportedly involved in the biosyntheses of these sugar residues were examined, rmlA, rmlB, wlaRA, wlaRB and wlaRG Specifically, these genes were cloned and expressed in Escherichia coli, and the corresponding enzymes were purified and tested for biochemical activity. Here we present data demonstrating that RmlA functions as a glucose-1-phosphate thymidylyltransferase and that RmlB is a thymidine diphosphate (dTDP)-glucose 4,6-dehydratase. We also show, through nuclear magnetic resonance spectroscopy and mass spectrometry analyses, that WlaRG, when utilized in coupled assays with either WlaRA or WlaRB and dTDP-4-keto-6-deoxyglucose, results in the production of either dTDP-3-amino-3,6-dideoxy-d-galactose (dTDP-Fuc3N) or dTDP-3-amino-3,6-dideoxy-d-glucose (dTDP-Qui3N), respectively. In addition, the X-ray crystallographic structures of the 3,4-ketoisomerases, WlaRA and WlaRB, were determined to 2.14 and 2.0 A resolutions, respectively. Taken together, the data reported herein demonstrate that C. jejuni 81116 utilizes five enzymes to synthesize dTDP-Fuc3N or dTDP-Qui3N and that WlaRG, an aminotransferase, can function on sugars with differing stereochemistry about their C-4' carbons. Importantly, the data reveal that C. jejuni 81116 has the ability to synthesize two isomeric ddHexN forms.

  Lipooligosaccharide, Campylobacter, quinovosamine, fucosamine, ketoisomerase

  NCBI PubMed ID: 28096310
  Publication DOI: 10.1093/glycob/cww136
  Journal NLM ID: 9104124
  Publisher: IRL Press at Oxford University Press
  Correspondence: Michel.Gilbert@nrc-cnrc.gc.ca
  Institutions: Department of Biochemistry, University of Wisconsin, Madison, WI, USA, National Research Council Canada, Human Health Therapeutics, 100 Sussex Drive, Ottawa, ON, Canada K1A 0R6
  Methods: 13C NMR, 1H NMR, X-ray, DNA techniques, genetic methods, biochemical methods, enzyme assay, CE-MS, cloning, crystallization
  
   
  
  Campylobacter jejuni 81116 (HS:6)
  CSDB ID 11936 (all data & tools)

Show legend Show as text

Structure type: monomer
Trivial name: TDP-6-deoxy-α-D-xylo-hex-4-ulose
Contained glycoepitopes: IEDB_227709

• Article ID: 3608
  Tello M, Rejzek M, Wilkinson B, Lawson DM, Field RA "Tyl1a, a TDP-6-deoxy-D-xylo-4-hexulose 3,4-isomerase from Streptomyces fradiae: structure prediction, mutagenesis and solvent isotope incorporation experiments to investigate reaction mechanism" - Chembiochem: a European Journal of Chemical Biology 9(8) (2008) 1295-1302
  

  Understanding the structure and mechanism of sugar nucleotide processing enzymes is invaluable in the generation of designer enzymes for biotransformation, for instance, in connection with engineering antibiotic glycosylation. In this study, homology modeling and mechanistic comparison to the structurally related RmlC epimerase family has been used to identify and assign functions to active-site residues in the Tyl1a-catalysed keto-sugar nucleotide isomerisation process. Tyl1a His63 is implicated as the base that initiates the isomerisation process by substrate C-3 deprotonation, with Arg109 stabilising the resulting enolate. Subsequent O-3 deprotonation (potentially by His65) and C-4 protonation (potentially by Tyr49) complete the isomerisation process

  Isomerases, sugar nucleotide, antibiotics, enzyme catalysis, structure analysis

  NCBI PubMed ID: 18425854
  Journal NLM ID: 100937360
  Publisher: Weinheim, Germany: Wiley Interscience
  Correspondence: david.lawson@bbsrc.ac.uk; rob.field@bbsrc.ac.uk
  Institutions: School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich, NR4 7TJ, UK
  Methods: 1H NMR, genetic methods, biochemical methods
  
   
  
  Streptomyces fradiae
  CSDB ID 23540 (all data & tools)

Show legend Show as text

Structure type: monomer
Trivial name: CDP-6-deoxy-D-xylo-hex-4-ulopyranose
Contained glycoepitopes: IEDB_141494

• Article ID: 3680
  Cunneen MM, De Castro C, Kenyon J, Parrilli M, Reeves PR, Molinaro A, Holst O, Skurnik M "The O-specific polysaccharide structure and biosynthetic gene cluster of Yersinia pseudotuberculosis serotype O:11" - Carbohydrate Research 344(12) (2009) 1533-1540
  

  In the Yersinia pseudotuberculosis serotyping scheme, 21 serotypes are present originating from about 30 different O-factors distributed within the species. With regard to the chemical structures of lipopolysaccharides (LPSs) and the genetic basis of their biosynthesis, a number, but not all, of Y. pseudotuberculosis strains representing different serotypes have been investigated. In order to present an overall picture of the relationship between genetics and structures, we have been working on the genetics and structures of various Y. pseudotuberculosis O-specific polysaccharides (OPSs). Here, we present a structural and genetic analysis of the Y. pseudotuberculosis serotype O:11 OPS. Our results showed that this OPS structure has the same backbone as that of Y. pseudotuberculosis O:1b, but with a 6d-l-Altf side-branch instead of Parf. The 3' end of the gene cluster is the same as that for O:1b and has the genes for synthesis of the backbone and for processing the completed repeat unit. The 5' end has genes for synthesis of 6d-l-Altf and its transfer to the repeating unit backbone. The pathway for the synthesis of the 6d-l-Altf appears to be different from that for 6d-l-Altp in Y. enterocolitica O:3. The chemical structure of the O:11 repeating unit is

  structure, O-specific polysaccharide, Yersinia pseudotuberculosis, O-specific polysaccharides, 6-Deoxy-L-altrofuranose, biosynthetic gene cluster

  NCBI PubMed ID: 19505680
  Journal NLM ID: 0043535
  Publisher: Elsevier
  Correspondence: oholst@fz-borstel.de (O. Holst)
  Institutions: Division of Microbiology, School of Molecular and Microbial Biosciences, University of Sydney, Australia
  Methods: 13C NMR, 1H NMR, methylation, NMR-2D, GC-MS, sugar analysis, NMR-1D, genetic methods
  
   
  
  Yersinia pseudotuberculosis O11
  CSDB ID 23985 (all data & tools)

Show legend Show as text

Structure type: polymer chemical repeating unit
Compound class: CPS
Contained glycoepitopes: IEDB_136906,IEDB_137472,IEDB_141794,IEDB_142488,IEDB_144998,IEDB_146664,IEDB_151528,IEDB_190606,IEDB_232584,IEDB_983931,SB_192,SB_7

• Article ID: 6454
  Sackett K, Brown P, Dutta K, Scully IL, Gangolli S, Looi K, Nemani S, Yu AYH, Kleven M, Xie J, Moran J, Pride MW, Anderson AS, Lotvin J "Identification of a Novel Keto Sugar Component in Streptococcus pneumoniae Serotype 12F Capsular Polysaccharide and Impact on Vaccine Immunogenicity" - Journal of Immunology 210(6) (2023) 764-773
  

  Implementation of conjugate vaccine technology revolutionized the ability to effectively elicit long-lasting immune responses to bacterial capsular polysaccharides. Although expansion of conjugate vaccine serotype coverage is designed to target residual disease burden to pneumococcal serotypes not contained in earlier vaccine versions, details of polysaccharide Ag structure, heterogeneity, and epitope structure components contributing to vaccine-mediated immunity are not always clear. Analysis of Streptococcus pneumoniae serotype 12F polysaccharide by two-dimensional nuclear magnetic resonance spectroscopy and mass spectrometry revealed a partial substitution of N-acetyl-galactosamine by the keto sugar 2-acetamido-2,6-dideoxy-xylo-hexos-4-ulose (Sug) in up to 25% of the repeat units. This substitution was not described in previous published structures for 12F. Screening a series of contemporary 12F strains isolated from humans (n = 17) identified Sug incorporation at varying levels in all strains examined. Thus, partial Sug substitution in S. pneumoniae serotype 12F may have always been present but is now detectable by state-of-the-art analytical techniques. During the steps of conjugation, the serotype 12F Sug epitope is modified by reduction, and both polysaccharide PPSV23 and conjugate PCV20 vaccines contain 12F Ags with little to no Sug epitope. Both PCV20 and PPSV23 vaccines were evaluated for protection against circulating 12F strains with varying amounts of Sug in their repeat unit based on an opsonophagocytic killing assay involving HL-60 cells and rabbit complement. Both vaccines elicited human-derived neutralizing Abs against serotype 12F, independent of Sug level between ∼2 and 25 mol%. These findings suggest that the newly identified serotype 12F Sug epitope is likely not an essential epitope for vaccine-elicited protection.

  Streptococcus pneumoniae, capsular polysaccharide, vaccine, serotype 12F

  NCBI PubMed ID: 36723033
  Publication DOI: 10.4049/jimmunol.2100849
  Journal NLM ID: 2985117R
  Publisher: Bethesda, MD: American Association of Immunologists
  Correspondence: J. Lotvin
  Institutions: Pfizer Analytical Research and Development, Groton, CT, Pfizer Analytical Research and Development, Chesterfield, MO, Pfizer Vaccine Research and Development, Pearl River, NY
  Methods: 13C NMR, 1H NMR, NMR-2D, LC-MS, ultrasonic treatment, LC-MS/MS, conjugation, opsonophagocytic assay, HILIC, genome sequencing, NaNH4 reduction
  
   
  
  Streptococcus pneumoniae 12F PFESP00919
  CSDB ID 26032 (all data & tools)