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Structure type: monomer
Trivial name: GDP-4-keto-3,6-dideoxymannose, GDP-4-keto-3,6-deoxymannose
Contained glycoepitopes: IEDB_141493,IEDB_149170,IEDB_190357

• Article ID: 3419
  Cook PD, Holden HM "A structural study of GDP-4-keto-6-deoxy-D-mannose-3-dehydratase: caught in the act of geminal diamine formation" - Biochemistry 46(49) (2007) 14215-14224
  

  Di- and trideoxysugars are an important class of carbohydrates synthesized by certain plants, fungi, and bacteria. Colitose, for example, is a 3,6-dideoxysugar found in the O-antigens of Gram-negative bacteria such as Escherichia coli, Salmonella enterica, Yersinia pseudotuberculosis, and Vibrio cholerae, among others. These types of dideoxysugars are thought to serve as antigenic determinants and to play key roles in bacterial defense and survival. Four enzymes are required for the biochemical synthesis of colitose starting from mannose-1-phosphate. The focus of this investigation, GDP-4-keto-6-deoxy-d-mannose-3-dehydratase (ColD), catalyzes the third step in the pathway, namely the PLP-dependent removal of the C3'-hydroxyl group from GDP-4-keto-6-deoxymannose. Whereas most PLP-dependent enzymes contain an active site lysine, ColD utilizes a histidine as its catalytic acid/base. The ping-pong mechanism of the enzyme first involves the conversion of PLP to PMP followed by the dehydration step. Here we present the three-dimensional structure of a site-directed mutant form of ColD whereby the active site histidine has been replaced with a lysine. The electron density reveals that the geminal diamine, a tetrahedral intermediate in the formation of PMP from PLP, has been trapped within the active site region. Functional assays further demonstrate that this mutant form of ColD cannot catalyze the dehydration reaction

  X-ray, Crystallography, Escherichia coli Proteins, GDP-4-keto-6-deoxy-d-mannose-3-dehydratase (ColD)

  NCBI PubMed ID: 17997582
  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, ESI-MS, genetic methods, biochemical methods, crystallization
  
   
  
  Escherichia coli O5a O55:H7
  CSDB ID 22014 (all data & tools)
• Article ID: 3453
  Cook PD, Carney AE, Holden HM "Accommodation of GDP-Linked Sugars in the Active Site of GDP-Perosamine Synthase" - Biochemistry 47(40) (2008) 10685-10693
  

  Perosamine (4-amino-4,6-dideoxy- d-mannose), or its N-acetylated form, is one of several dideoxy sugars found in the O-antigens of such infamous Gram-negative bacteria as Vibrio cholerae O1 and Escherichia coli O157:H7. It is added to the bacterial O-antigen via a nucleotide-linked version, namely GDP-perosamine. Three enzymes are required for the biosynthesis of GDP-perosamine starting from mannose 1-phosphate. The focus of this investigation is GDP-perosamine synthase from Caulobacter crescentus, which catalyzes the final step in GDP-perosamine synthesis, the conversion of GDP-4-keto-6-deoxymannose to GDP-perosamine. The enzyme is PLP-dependent and belongs to the aspartate aminotransferase superfamily. It contains the typically conserved active site lysine residue, which forms a Schiff base with the PLP cofactor. Two crystal structures were determined for this investigation: a site-directed mutant protein (K186A) complexed with GDP-perosamine and the wild-type enzyme complexed with an unnatural ligand, GDP-3-deoxyperosamine. These structures, determined to 1.6 and 1.7 A resolution, respectively, revealed the manner in which products, and presumably substrates, are accommodated within the active site pocket of GDP-perosamine synthase. Additional kinetic analyses using both the natural and unnatural substrates revealed that the K m for the unnatural substrate was unperturbed relative to that of the natural substrate, but the k cat was lowered by a factor of approximately 200. Taken together, these studies shed light on why GDP-perosamine synthase functions as an aminotransferase whereas another very similar PLP-dependent enzyme, GDP-4-keto-6-deoxy- d-mannose 3-dehydratase or ColD, catalyzes a dehydration reaction using the same substrate.

  biosynthesis, O-antigen, Escherichia coli O157:H7, Vibrio cholerae O1, perosamine, synthase

  NCBI PubMed ID: 18795799
  Journal NLM ID: 0370623
  Publisher: American Chemical Society
  Correspondence: Hazel_Holden@biochem.wisc.edu
  Institutions: Department of Biochemistry, UniVersity of Wisconsin, Madison, Wisconsin 53706
  Methods: X-ray, sugar analysis, genetic methods, biochemical methods, HPLC, crystallization
  
   
  
  Escherichia coli O157:H7, Vibrio cholerae O1
  CSDB ID 22864 (all data & tools)
• Article ID: 3454
  Cook PD, Holden HM "GDP-Perosamine Synthase: Structural Analysis and Production of a Novel Trideoxysugar" - Biochemistry 47(9) (2008) 2833-2840
  

  Perosamine or 4-amino-4,6-dideoxy- d-mannose is an unusual sugar found in the O-antigens of some Gram-negative bacteria such as Vibrio cholerae O1 (the causative agent of cholera) or Escherichia coli O157:H7 (the leading cause of food-borne illnesses). It and similar deoxysugars are added to the O-antigens of bacteria via the action of glycosyltransferases that employ nucleotide-linked sugars as their substrates. The focus of this report is GDP-perosamine synthase, a PLP-dependent enzyme that catalyzes the last step in the formation of GDP-perosamine, namely, the amination of the sugar C-4'. Here we describe the three-dimensional structure of the enzyme from Caulobacter crescentus determined to a nominal resolution of 1.8 A and refined to an R-factor of 17.9%. The overall fold of the enzyme places it into the well-characterized aspartate aminotransferase superfamily. Each subunit of the dimeric enzyme contains a seven-stranded mixed beta-sheet, a two-stranded antiparallel beta-sheet, and 12 alpha-helices. Amino acid residues from both subunits form the active sites of the GDP-perosamine synthase dimer. Recently, the structure of another PLP-dependent enzyme, GDP-4-keto-6-deoxy- d-mannose-3-dehydratase (or ColD), was determined in our laboratory, and this enzyme employs the same substrate as GDP-perosamine synthase. Unlike GDP-perosamine synthase, however, ColD functions as a dehydratase that removes the sugar C-3' hydroxyl group. By purifying the ColD product and reacting it with purified GDP-perosamine synthase, we have produced a novel GDP-linked sugar, GDP-4-amino-3,4,6-trideoxy-d-mannose. Details describing the X-ray structural investigation of GDP-perosamine synthase and the enzymatic synthesis of GDP-4-amino-3,4,6-trideoxy-d-mannose are presented.

  X-ray, Escherichia coli O157:H7, O-antigens, glycosyltransferases, Vibrio cholerae O1, perosamine, enzymatic synthesis

  NCBI PubMed ID: 18247575
  Journal NLM ID: 0370623
  Publisher: American Chemical Society
  Correspondence: Hazel_Holden@biochem.wisc.edu
  Institutions: Department of Biochemistry, UniVersity of Wisconsin, Madison, Wisconsin 53706
  Methods: 13C NMR, 1H NMR, X-ray, sugar analysis, ESI-MS, genetic methods, biochemical methods, HPLC, crystallization
  
   
  
  Caulobacter crescentus CB15
  CSDB ID 22872 (all data & tools)
• Article ID: 3455
  Cook PD, Holden HM "GDP-4-keto-6-deoxy-D-mannose-3-dehydratase: Accommodating a sugar substrate in the active site" - Journal of Biological Chemistry 283(7) (2008) 4295-4303
  

  Colitose is a dideoxysugar found in the O-antigen of the lipopolysaccharide that coats the outer membrane of some Gram-negative bacteria. Four enzymes are required for its production starting from D-mannose-1-phosphate and GTP. The focus of this investigation is GDP-4-keto-6-deoxy-D-mannose-3-dehydratase or ColD, which catalyzes the removal of the C3'-hydroxyl group from GDP-4-keto-6-deoxymannose. The enzyme is PLP-dependent, but unlike most of these proteins, the conserved lysine residue which covalently holds the cofactor in the active site is replaced with a histidine residue. Here we describe the three-dimensional structure of ColD determined to 1.7 A resolution whereby the active site histidine has been replaced with an asparagine residue. For this investigation, crystals of the site-directed mutant protein were grown in the presence of GDP-4-amino-4,6-dideoxy-D-mannose (GDP-perosamine). The electron density map clearly reveals the presence of the sugar analog trapped in the active site as an external aldimine. The active site is positioned between the two subunits of the dimer. Whereas the pyrophosphoryl groups of the ligand are anchored to the protein via Arg 219 and Arg 331, the hydroxyl groups of the hexose only lie within hydrogen bonding distance to ordered water molecules. Interestingly, the hexose moiety of the ligand adopts a boat rather than the typically observed chair conformation. Activity assays demonstrate that this mutant protein cannot catalyze the dehydration step. Additionally, we report data revealing that wild-type ColD is able to catalyze the production of GDP-4-keto-3,6-dideoxy-mannose using GDP-perosamine instead of GDP-4-keto-6-deoxymannose as a substrate.

  Enzymes, colitose, ColD, dehydratase

  NCBI PubMed ID: 18045869
  Journal NLM ID: 2985121R
  Publisher: Baltimore, MD: American Society for Biochemistry and Molecular Biology
  Correspondence: Hazel_Holden@biochem.wisc.edu
  Institutions: Department of Biochemistry, University of Wisconsin, Madison, WI, USA
  Methods: X-ray, sugar analysis, ESI-MS, genetic methods, biochemical methods, HPLC, crystallization
  
   
  
  Escherichia coli O55:H7
  CSDB ID 22879 (all data & tools)

Show legend Show as text

Structure type: monomer
Trivial name: GDP-3,6-dideoxy-D-threo-hexos-4-ulose
Contained glycoepitopes: IEDB_141493,IEDB_149170,IEDB_190357

• Article ID: 4362
  Ovchinnikova OG, Liu B, Guo D, Kocharova NA, Shashkov AS, Chen M, Feng L, Rozalski A, Knirel YA, Wang L "Localization and molecular characterization of putative O-antigen gene clusters of Providencia species" - Microbiology 158(4) (2012) 1024-1036
  

  Enterobacteria of the genus Providencia are opportunistic human pathogens associated with urinary tract and wound infections, as well as enteric diseases. The lipopolysaccharide (LPS) O antigen confers major antigenic variability upon the cell surface and is used for serotyping of Gram-negative bacteria. Recently, Providencia O antigen structures have been extensively studied, but no data on the location and organization of the O antigen gene cluster have been reported. In this study, the four Providencia genome sequences available were analysed, and the putative O antigen gene cluster was identified in the polymorphic locus between the cpxA and yibK genes. This finding provided the necessary information for designing primers, and cloning and sequencing the O antigen gene clusters from five more Providencia alcalifaciens strains. The gene functions predicted in silico were in agreement with the known O antigen structures; furthermore, annotation of the genes involved in the three-step synthesis of GDP-colitose (gmd, colD and colC) was supported by cloning and biochemical characterization of the corresponding enzymes. In one strain (P. alcalifaciens O39), no polysaccharide product of the gene cluster in the cpxA-yibK locus was found, and hence genes for synthesis of the existing O antigen are located elsewhere in the genome. In addition to the putative O antigen synthesis genes, homologues of wza, wzb, wzc and (in three strains) wzi, required for the surface expression of capsular polysaccharides, were found upstream of yibK in all species except Providencia rustigianii, suggesting that the LPS of these species may be attributed to the so-called K LPS (K(LPS)). The data obtained open a way for development of a PCR-based typing method for identification of Providencia isolates.

  Lipopolysaccharide, Providencia alcalifaciens, O-antigen gene cluster, O-Polysaccharide structure

  NCBI PubMed ID: 22282517
  Publication DOI: 10.1099/mic.0.055210-0
  Journal NLM ID: 0376646
  Publisher: Washington, DC: Kluwer Academic/Plenum Publishers
  Correspondence: wanglei@nankai.edu.cn; yknirel@gmail.com
  Institutions: TEDA School of Biological Sciences and Biotechnology, Nankai University, 23 Hongda Street, TEDA, 300457 Tianjin, PR China
  Methods: 13C NMR, 1H NMR, NMR-2D, PCR, DNA sequencing, SDS-PAGE, glycosyltransferase assays, 31P NMR, ESI-MS, genetic methods, HPLC
  
   
  
  Providencia alcalifaciens O39, Providencia alcalifaciens O6
  CSDB ID 28538 (all data & tools)