Show legend Show as text

Structure type: monomer
Trivial name: UDP-2-acetamido-2-deoxy-D-ribo-hex-3-uluronic acid, UDP-2-aceamido-2-deoxy-α-D-ribo-hex-3-uluronic acid, UDP-3-keto-GlcNAcA, UDP-GlcNAc(3keto)A

• Article ID: 3320
  Westman EL, McNally DJ, Rejzek M, Miller WL, Kannathasan VS, Preston A, Maskell DJ, Field RA, Brisson JR, Lam JS "Identification and biochemical characterization of two novel UDP-2,3-diacetamido-2,3-dideoxy-α-D-glucuronic acid 2-epimerases from respiratory pathogens" - Biochemical Journal 405(1) (2007) 123-130
  

  The heteropolymeric O-antigen of the lipopolysaccharide from Pseudomonas aeruginosa serogroup O5 as well as the band-A trisaccharide from Bordetella pertussis contain the di-N-acetylated mannosaminuronic acid derivative, β-D-ManNAc3NAcA (2,3-diacetamido-2,3-dideoxy-β-D-mannuronic acid). The biosynthesis of the precursor for this sugar is proposed to require five steps, through which UDP-α-D-GlcNAc (UDP-N-acetyl-α-D-glucosamine) is converted via four steps into UDP-α-D-GlcNAc3NAcA (UDP-2,3-diacetamido-2,3-dideoxy-α-D-glucuronic acid), and this intermediate compound is then epimerized by WbpI (P. aeruginosa), or by its orthologue, WlbD (B. pertussis), to form UDP-α-D-ManNAc3NAcA (UDP-2,3-diacetamido-2,3-dideoxy-α-D-mannuronic acid). UDP-α-D-GlcNAc3NAcA, the proposed substrate for WbpI and WlbD, was obtained through chemical synthesis. His6-WbpI and His6-WlbD were overexpressed and then purified by affinity chromatography using FPLC. Capillary electrophoresis was used to analyse reactions with each enzyme, and revealed that both enzymes used UDP-α-D-GlcNAc3NAcA as a substrate, and reacted optimally in sodium phosphate buffer (pH 6.0). Neither enzyme utilized UDP-α-D-GlcNAc, UDP-α-D-GlcNAcA (UDP-2-acetamido-2,3-dideoxy-α-D-glucuronic acid) or UDP-α-D-GlcNAc3NAc (UDP-2,3-diacetamido-2,3-dideoxy-α-D-glucose) as substrates. His6-WbpI or His6-WlbD reactions with UDP-α-D-GlcNAc3NAcA produce a novel peak with an identical retention time, as shown by capillary electrophoresis. To unambiguously characterize the reaction product, enzyme-substrate reactions were allowed to proceed directly in the NMR tube and conversion of substrate into product was monitored over time through the acquisition of a proton spectrum at regular intervals. Data collected from one- and two-dimensional NMR experiments showed that His6-WbpI catalysed the 2-epimerization of UDP-α-D-GlcNAc3NAcA, converting it into UDP-α-D-ManNAc3NAcA. Collectively, these results provide evidence that WbpI and WlbD are UDP-2,3-diacetamido-2,3-dideoxy-α-D-glucuronic acid 2-epimerases

  Lipopolysaccharide, O-antigen, Pseudomonas aeruginosa, 3-diacetamido-2, 2-epimerase, mannosaminuronic acid biosynthesis, sugar-nucleotide metabolism, UDP-2, 3-dideoxy-α-D-glucuronic acid

  NCBI PubMed ID: 17346239
  Publication DOI: 10.1042/BJ20070017
  Journal NLM ID: 2984726R
  Publisher: London, UK : Published by Portland Press on behalf of the Biochemical Society
  Correspondence: jlam@uoguelph.ca
  Institutions: Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada, N1G 2W1
  Methods: 13C NMR, 1H NMR, 31P NMR, NMR-1D, genetic methods, biochemical methods, HPLC, capillary electrophoresis (CE), IR-MALDI-TOF MS
  
   
  
  Pseudomonas aeruginosa O5
  CSDB ID 21683 (all data & tools)
• Article ID: 3569
  Westman EL, Preston A, Field RA, Lam JS "Biosynthesis of a rare di-N-acetylated sugar in the lipopolysaccharide of both Pseudomonas aeruginosa and Bordetella pertussis occurs via an identical scheme despite different gene clusters" - Journal of Bacteriology 190(18) (2008) 6060-6069
  

  Pseudomonas aeruginosa and Bordetella pertussis produce lipopolysaccharide (LPS) that contains 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid (D-ManNAc3NAcA). A five-enzyme biosynthetic pathway that requires WbpA, WbpB, WbpE, WbpD and WbpI has been proposed for the production of this sugar in P. aeruginosa, based on analysis of genes present in the B-band LPS biosynthesis cluster. In the analogous B. pertussis cluster, homologs of wbpB-I are present but a putative dehydrogenase was missing; therefore, the biosynthetic mechanism for UDP-D-ManNAc3NAcA was unclear. Non-polar knockout mutants of each P. aeruginosa gene were constructed. Complementation analysis of the mutants demonstrated that B-band LPS production was restored to P. aeruginosa knockout mutants when the relevant B. pertussis genes were supplied in trans. Thus, the genes that encode the putative oxidase, transaminase, N-acetyltransferase, and epimerase enzymes in B. pertussis are functional homologs of those in P. aeruginosa. Two candidate dehydrogenase genes were located by searching the B. pertussis genome; these have 80% identity to P. aeruginosa wbpO (serotype O6) and 32% identity to wbpA (serotype O5). These genes, wbpO1629 and wbpO3150, were shown to complement a wbpA knockout in P. aeruginosa. Capillary electrophoresis was used to characterize the enzymatic activities of purified WbpO1629 and WbpO3150, and mass spectrometry analysis confirmed that the two enzymes are dehydrogenases capable of converting UDP-D-GlcNAc, to a lesser extent UDP-D-GalNAc, and to a much lesser extent, UDP-D-Glc. Together, these results suggest that B. pertussis produces UDP-D-ManNAc3NAcA through the same pathway proposed in P. aeruginosa, despite differences in the genomic context of the genes involved

  Lipopolysaccharide, biosynthesis, Pseudomonas aeruginosa, gene cluster, Bordetella pertussis, WbpA, WbpI

  NCBI PubMed ID: 18621892
  Journal NLM ID: 2985120R
  Publisher: American Society for Microbiology
  Correspondence: jlam@uoguelph.ca
  Institutions: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada, School of Clinical Veterinary Science, University of Bristol, Langford, Bristol, UK, Department of Biological Chemistry, John Innes Centre, Norwich, UK
  Methods: MALDI-MS, Western blotting, genetic methods, biochemical methods, capillary electrophoresis (CE)
  
   
  
  Pseudomonas aeruginosa, Bordetella pertussis
  CSDB ID 23330 (all data & tools)
• Article ID: 3803
  Westman EL, McNally DJ, Charchoglyan A, Brewer D, Field RA, Lam JS "Characterization of WbpB, WbpE, and WbpD, and reconstitution of a pathway for the biosynthesis of UDP-2,3-diacetamido-2,3-dideoxy-D-mannuronic acid in Pseudomonas aeruginosa" - Journal of Biological Chemistry 284(18) (2009) 11854-11862
  

  The lipopolysaccharide of Pseudomonas aeruginosa PAO1 contains an unusual sugar, 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid (D-ManNAc3NAcA). wbpB, wbpE, and wbpD are thought to encode oxidase, transaminase, and N-acetyltransferase enzymes. To characterize their functions, recombinant proteins were overexpressed and purified from heterologous hosts. Activities of His6-WbpB and His6-WbpE were detected only when both proteins were combined in the same reaction. Using a direct MALDI-TOF mass spectrometry approach, we identified ions that corresponded to the predicted products of WbpB (UDP-3-keto-D-GlcNAcA) and WbpE (UDP-D-GlcNAc3NA) in the coupled enzyme-substrate reaction. Additionally, in reactions involving WbpB, WbpE and WbpD, an ion consistent with the expected product of WbpD (UDP-D-GlcNAc3NAcA) was identified. Preparative quantities of UDP-D-GlcNAc3NA and UDP-D-GlcNAc3NAcA were enzymatically synthesized. These compounds were purified by HPLC and their structures were elucidated by NMR spectroscopy. This is the first report of the functional characterization of these proteins, and the enzymatic synthesis of UDP-D-GlcNAc3NA and UDP-D-GlcNAc3NAcA.

  biosynthesis, Pseudomonas aeruginosa, MALDI-TOF mass spectrometry, enzymatic synthesis, Recombinant Proteins

  NCBI PubMed ID: 19282284
  Journal NLM ID: 2985121R
  Publisher: Baltimore, MD: American Society for Biochemistry and Molecular Biology
  Correspondence: jlam@uoguelph.ca
  Institutions: Microbiology, University of Guelph, Guelph, ON N1G 2W1
  Methods: 13C NMR, 1H NMR, SDS-PAGE, 31P NMR, MALDI-TOF MS, NMR-1D, genetic methods, biochemical methods, capillary electrophoresis (CE)
  
   
  
  Pseudomonas aeruginosa PAO1
  CSDB ID 24261 (all data & tools)
• Article ID: 3885
  Larkin A, Olivier NB, Imperiali B "Structural Analysis of WbpE from Pseudomonas aeruginosa PAO1: A Nucleotide Sugar Aminotransferase Involved in O-antigen Assembly" - Biochemistry 49(33) (2010) 7227-7237
  

  In recent years, the opportunistic pathogen Pseudomonas aeruginosa has emerged as a major source of hospital-acquired infections. Effective treatment has proven increasingly difficult due to the spread of multidrug resistant strains and thus requires a deeper understanding of the biochemical mechanisms of pathogenicity. The central carbohydrate of the P. aeruginosa PAO1 (O5) B-band O-antigen, ManNAc(3NAc)A, has been shown to be critical for virulence and is produced in a stepwise manner by five enzymes in the Wbp pathway (WbpA, WbpB, WbpE, WbpD and WbpI). Herein, we present the crystal structure of the aminotransferase WbpE from P. aeruginosa PAO1 in complex with the cofactor pyridoxal 5'-phosphate (PLP) and product UDP-GlcNAc(3NH2)A as the external aldimine at 1.9 A resolution. We also report the structures of WbpE in complex with PMP alone as well as the PLP internal aldimine, and show that the dimeric structure of WbpE observed in the crystal structure is confirmed by analytical ultracentrifugation. Analysis of these structures reveals that the active site of the enzyme is composed of residues from both subunits. In particular, we show that a key residue (Arg229), which has previously been implicated in direct interactions with the carboxylate moiety of α-ketoglutarate, is also uniquely positioned to bestow specificity for the 6' carboxyl group of GlcNAc(3NH2)A through a salt bridge. This finding is intriguing, because while an analogous basic residue is present in WbpE homologs that do not process C6'-carboxyl-modified saccharides, recent structural studies reveal that this side chain is retracted to accommodate a neutral C-6' carbon. This work represents the first structural analysis of a nucleotide sugar aminotransferase with a bound product modified at the C2', C3', and C6' positions and provides insight into a novel target for treatment of P. aeruginosa infection.

  O-antigen, Pseudomonas aeruginosa, crystal structure, WbpA, WbpI, aminotransferase WbpE

  NCBI PubMed ID: 20604544
  Journal NLM ID: 0370623
  Publisher: American Chemical Society
  Correspondence: imper@mit.edu
  Institutions: Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.
  Methods: SDS-PAGE, MALDI-MS, Western blotting, genetic methods, biochemical methods, crystallization
  
   
  
  Pseudomonas aeruginosa PAO1 (05)
  CSDB ID 25732 (all data & tools)
• Article ID: 3954
  Thoden JB, Holden HM "Structural and functional studies of WlbA: A dehydrogenase involved in the biosynthesis of 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid" - Biochemistry 49(36) (2010) 7939-7948
  

  2,3-Diacetamido-2,3-dideoxy-d-mannuronic acid (ManNAc3NAcA) is an unusual dideoxy sugar first identified nearly 30 years ago in the lipopolysaccharide of Pseudomonas aeruginosa O:3a,d. It has since been observed in other organisms, including Bordetella pertussis, the causative agent of whooping cough. Five enzymes are required for the biosynthesis of UDP-ManNAc3NAcA starting from UDP-N-acetyl-d-glucosamine. Here we describe a structural study of WlbA, the NAD-dependent dehydrogenase that catalyzes the second step in the pathway, namely, the oxidation of the C-3' hydroxyl group on the UDP-linked sugar to a keto moiety and the reduction of NAD(+) to NADH. This enzyme has been shown to use α-ketoglutarate as an oxidant to regenerate the oxidized dinucleotide. For this investigation, three different crystal structures were determined: the enzyme with bound NAD(H), the enzyme in a complex with NAD(H) and α-ketoglutarate, and the enzyme in a complex with NAD(H) and its substrate (UDP-N-acetyl-d-glucosaminuronic acid). The tetrameric enzyme assumes an unusual quaternary structure with the dinucleotides positioned quite closely to one another. Both α-ketoglutarate and the UDP-linked sugar bind in the WlbA active site with their carbon atoms (C-2 and C-3', respectively) abutting the re face of the cofactor. They are positioned approximately 3 A from the nicotinamide C-4. The UDP-linked sugar substrate adopts a highly unusual curved conformation when bound in the WlbA active site cleft. Lys 101 and His 185 most likely play key roles in catalysis.

  biosynthesis, Pseudomonas, Bordetella, Bordetella pertussis, crystal structure, Enzymes, dehydrogenase, WlbA

  NCBI PubMed ID: 20690587
  Journal NLM ID: 0370623
  Publisher: American Chemical Society
  Correspondence: Hazel_Holden@biochem.wisc.edu
  Institutions: Department of Biochemistry, University of Wisconsin, Madison, WI, USA
  Methods: X-ray, genetic methods, biochemical methods, crystallization
  
   
  
  Pseudomonas aeruginosa PAO1-LAC, Thermus thermophilus (ATCC BAA-163)
  CSDB ID 25867 (all data & tools)