Dataset Information

Crystal structure of TDP-fucosamine acetyltransferase (WecD) from Escherichia coli, an enzyme required for enterobacterial common antigen synthesis.

ABSTRACT: Enterobacterial common antigen (ECA) is a polysaccharide found on the outer membrane of virtually all gram-negative enteric bacteria and consists of three sugars, N-acetyl-d-glucosamine, N-acetyl-d-mannosaminuronic acid, and 4-acetamido-4,6-dideoxy-d-galactose, organized into trisaccharide repeating units having the sequence -->3)-alpha-d-Fuc4NAc-(1-->4)-beta-d-ManNAcA-(1-->4)-alpha-d-GlcNAc-(1-->. While the precise function of ECA is unknown, it has been linked to the resistance of Shiga-toxin-producing Escherichia coli (STEC) O157:H7 to organic acids and the resistance of Salmonella enterica to bile salts. The final step in the synthesis of 4-acetamido-4,6-dideoxy-d-galactose, the acetyl-coenzyme A (CoA)-dependent acetylation of the 4-amino group, is carried out by TDP-fucosamine acetyltransferase (WecD). We have determined the crystal structure of WecD in apo form at a 1.95-Angstrom resolution and bound to acetyl-CoA at a 1.66-Angstrom resolution. WecD is a dimeric enzyme, with each monomer adopting the GNAT N-acetyltransferase fold, common to a number of enzymes involved in acetylation of histones, aminoglycoside antibiotics, serotonin, and sugars. The crystal structure of WecD, however, represents the first structure of a GNAT family member that acts on nucleotide sugars. Based on this cocrystal structure, we have used flexible docking to generate a WecD-bound model of the acetyl-CoA-TDP-fucosamine tetrahedral intermediate, representing the structure during acetyl transfer. Our structural data show that WecD does not possess a residue that directly functions as a catalytic base, although Tyr208 is well positioned to function as a general acid by protonating the thiolate anion of coenzyme A.

SUBMITTER: Hung MN

PROVIDER: S-EPMC1540030 | biostudies-literature | 2006 Aug

REPOSITORIES: biostudies-literature

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Crystal structure of TDP-fucosamine acetyltransferase (WecD) from Escherichia coli, an enzyme required for enterobacterial common antigen synthesis.

Hung Ming-Ni MN Rangarajan Erumbi E Munger Christine C Nadeau Guy G Sulea Traian T Matte Allan A

Journal of bacteriology 20060801 15

Enterobacterial common antigen (ECA) is a polysaccharide found on the outer membrane of virtually all gram-negative enteric bacteria and consists of three sugars, N-acetyl-d-glucosamine, N-acetyl-d-mannosaminuronic acid, and 4-acetamido-4,6-dideoxy-d-galactose, organized into trisaccharide repeating units having the sequence -->3)-alpha-d-Fuc4NAc-(1-->4)-beta-d-ManNAcA-(1-->4)-alpha-d-GlcNAc-(1-->. While the precise function of ECA is unknown, it has been linked to the resistance of Shiga-toxin- ...[more]

PMID: 16855251

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Project description:The polysaccharide chains of enterobacterial common antigen (ECA) are comprised of the trisaccharide repeat unit Fuc4NAc-ManNAcA-GlcNAc, where Fuc4NAc is 4-acetamido-4,6-dideoxy-D-galactose, ManNAcA is N-acetyl-D-mannosaminuronic acid, and GlcNAc is N-acetyl-D-glucosamine. Individual trisaccharide repeat units are assembled as undecaprenyl-linked intermediates in a sequence of reactions that culminate in the transfer of Fuc4NAc from TDP-Fuc4NAc to ManNAcA-GlcNAc-pyrophosphorylundecaprenol (lipid II) to yield Fuc4NAc-ManNAcA-GlcNAc-pyrophosphorylundecaprenol (lipid III), the donor of trisaccharide repeat units for ECA polysaccharide chain elongation. Most of the genes known to be involved in ECA assembly are located in the wec gene cluster located at ca. 85.4 min on the Escherichia coli chromosome. The available data suggest that the structural gene for the TDP-Fuc4NAc:lipid II Fuc4NAc transferase also resides in the wec gene cluster; however, the location of this gene has not been unequivocally defined. Previous characterization of the nucleotide sequence of the wec gene cluster in the region between o416 and wecG revealed that it contained three open reading frames: o74, o204, and o450. In contrast, the results of experiments described in the current investigation revealed that it contains only two open reading frames, o359 and o450. Mutants of E. coli possessing null mutations in o359 were unable to synthesize ECA, and they accumulated lipid II. In addition, the in vitro incorporation of [(3)H]FucNAc from TDP-[(3)H]Fuc4NAc into lipid II was not observed in reaction mixtures using cell extracts obtained from these mutants as a source of enzyme. The ECA-negative phenotype of these mutants was complemented by plasmid constructs containing the wild-type o359 allele, and Fuc4NAc transferase activity was demonstrated by using cell extracts obtained from the complemented mutants. Furthermore, partially purified o359 gene product, expressed as recombinant C-terminal His-tagged protein, was able to catalyze the in vitro transfer of [(3)H]Fuc4NAc from TDP-[(3)H]Fuc4NAc to lipid II. Our data support the conclusion that o359 of the wec gene cluster of E. coli is the structural gene for the TDP-Fuc4NAc:lipid II Fuc4NAc transferase involved in the synthesis ECA trisaccharide repeat units.

Project description:The Gram-negative cell envelope is a complex structure delineating the cell from its environment. Recently, we found that enterobacterial common antigen (ECA) plays a role maintaining the outer membrane (OM) permeability barrier, which excludes toxic molecules including many antibiotics. ECA is a conserved carbohydrate found throughout Enterobacterales (e.g., Salmonella, Klebsiella, and Yersinia). There are two OM forms of ECA (phosphoglyceride-linked ECAPG and lipopolysaccharide-linked ECALPS) and one periplasmic form of ECA (cyclic ECACYC). ECAPG, found in the outer leaflet of the OM, consists of a linear ECA oligomer attached to phosphoglyceride through a phosphodiester linkage. The process through which ECAPG is produced from polymerized ECA is unknown. Therefore, we set out to identify genes interacting genetically with ECAPG biosynthesis in Escherichia coli K-12 using the competition between ECA and peptidoglycan biosynthesis. Through transposon-directed insertion sequencing, we identified an interaction between elyC and ECAPG biosynthesis. ElyC is an inner membrane protein previously shown to alter peptidoglycan biosynthesis rates. We found ΔelyC was lethal specifically in strains producing ECAPG without other ECA forms, suggesting ECAPG biosynthesis impairment or dysregulation. Further characterization suggested ElyC inhibits ECAPG synthesis in a posttranscriptional manner. Moreover, the full impact of ElyC on ECA levels requires the presence of ECACYC. Our data demonstrate ECACYC can regulate ECAPG synthesis in strains wild type for elyC. Overall, our data demonstrate ElyC and ECACYC act in a novel pathway that regulates the production of ECAPG, supporting a model in which ElyC provides feedback regulation of ECAPG production based on the periplasmic levels of ECACYC. IMPORTANCE Enterobacterial common antigen (ECA) is a conserved polysaccharide present on the surface of the outer membrane (OM) and in the periplasm of the many pathogenic bacteria belonging to Enterobacterales, including Klebsiella pneumoniae, Salmonella enterica, and Yersinia pestis. As the OM is a permeability barrier that excludes many antibiotics, synthesis pathways for OM molecules are promising targets for antimicrobial discovery. Here, we elucidated, in E. coli K-12, a new pathway for the regulation of biosynthesis of one cell surface form of ECA, ECAPG. In this pathway, an inner membrane protein, ElyC, and the periplasmic form of ECA, ECACYC, genetically interact to inhibit the synthesis of ECAPG, potentially through feedback regulation based on ECACYC levels. This is the first insight into the pathway responsible for synthesis of ECAPG and represents a potential target for weakening the OM permeability barrier. Furthermore, this pathway provides a tool for experimental manipulation of ECAPG levels.

Dataset Information

Crystal structure of TDP-fucosamine acetyltransferase (WecD) from Escherichia coli, an enzyme required for enterobacterial common antigen synthesis.

Publications

Crystal structure of TDP-fucosamine acetyltransferase (WecD) from Escherichia coli, an enzyme required for enterobacterial common antigen synthesis.

Similar Datasets

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