scholarly journals Crystal Structure of TDP-Fucosamine Acetyltransferase (WecD) from Escherichia coli, an Enzyme Required for Enterobacterial Common Antigen Synthesis

2006 ◽  
Vol 188 (15) ◽  
pp. 5606-5617 ◽  
Author(s):  
Ming-Ni Hung ◽  
Erumbi Rangarajan ◽  
Christine Munger ◽  
Guy Nadeau ◽  
Traian Sulea ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Coenzyme A ◽  
Structural Data ◽  
Enteric Bacteria ◽  
Common Antigen ◽  
Acetyl Coa ◽  
Enterobacterial Common Antigen ◽  
Thiolate Anion ◽  
Acetyl Transfer

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)-α-d-Fuc4NAc-(1→4)-β-d-ManNAcA-(1→4)-α-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-Å resolution and bound to acetyl-CoA at a 1.66-Å 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.

scholarly journals Lipopolysaccharide-Linked Enterobacterial Common Antigen (ECALPS) Occurs in Rough Strains of Escherichia coli R1, R2, and R4

2020 ◽  
Vol 21 (17) ◽  
pp. 6038
Author(s):  
Anna Maciejewska ◽  
Marta Kaszowska ◽  
Wojciech Jachymek ◽  
Czeslaw Lugowski ◽  
Jolanta Lukasiewicz
Keyword(s):  
Escherichia Coli ◽  
Surface Antigen ◽  
Structural Data ◽  
Outer Core ◽  
Permeability Barrier ◽  
Common Antigen ◽  
Enterobacterial Common Antigen ◽  
Specific Polysaccharide ◽  
K 12 ◽  
Outer Membrane Permeability

Enterobacterial common antigen (ECA) is a conserved surface antigen characteristic for Enterobacteriaceae. It is consisting of trisaccharide repeating unit, →3)-α-d-Fucp4NAc-(1→4)-β-d-ManpNAcA-(1→4)-α-d-GlcpNAc-(1→, where prevailing forms include ECA linked to phosphatidylglycerol (ECAPG) and cyclic ECA (ECACYC). Lipopolysaccharide (LPS)-associated form (ECALPS) has been proved to date only for rough Shigella sonnei phase II. Depending on the structure organization, ECA constitutes surface antigen (ECAPG and ECALPS) or maintains the outer membrane permeability barrier (ECACYC). The existence of LPS was hypothesized in the 1960–80s on the basis of serological observations. Only a few Escherichia coli strains (i.e., R1, R2, R3, R4, and K-12) have led to the generation of anti-ECA antibodies upon immunization, excluding ECAPG as an immunogen and conjecturing ECALPS as the only immunogenic form. Here, we presented a structural survey of ECALPS in E. coli R1, R2, R3, and R4 to correlate previous serological observations with the presence of ECALPS. The low yields of ECALPS were identified in the R1, R2, and R4 strains, where ECA occupied outer core residues of LPS that used to be substituted by O-specific polysaccharide in the case of smooth LPS. Previously published observations and hypotheses regarding the immunogenicity and biosynthesis of ECALPS were discussed and correlated with presented herein structural data.

scholarly journals Identification and Biosynthesis of Cyclic Enterobacterial Common Antigen in Escherichia coli

2003 ◽  
Vol 185 (6) ◽  
pp. 1995-2004 ◽  
Author(s):  
Paul J. A. Erbel ◽  
Kathleen Barr ◽  
Ninguo Gao ◽  
Gerrit J. Gerwig ◽  
Paul D. Rick ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Repeat Unit ◽  
Enteric Bacteria ◽  
Water Soluble ◽  
Ionization Mass ◽  
Common Antigen ◽  
Esi Ms ◽  
Enterobacterial Common Antigen ◽  
Repeat Units ◽  
K 12

ABSTRACT Phosphoglyceride-linked enterobacterial common antigen (ECAPG) is a cell surface glycolipid that is synthesized by all gram-negative enteric bacteria. The carbohydrate portion of ECAPG consists of linear heteropolysaccharide chains 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. The potential reducing terminal GlcNAc residue of each polysaccharide chain is linked via phosphodiester linkage to a phosphoglyceride aglycone. We demonstrate here the occurrence of a water-soluble cyclic form of enterobacterial common antigen, ECACYC, purified from Escherichia coli strains B and K-12 with solution nuclear magnetic resonance (NMR) spectroscopy, electrospray ionization mass spectrometry (ESI-MS), and additional biochemical methods. The ECACYC molecules lacked an aglycone and contained four trisaccharide repeat units that were nonstoichiometrically substituted with up to four O-acetyl groups. ECACYC was not detected in mutant strains that possessed null mutations in the wecA, wecF, and wecG genes of the wec gene cluster. These observations corroborate the structural data obtained by NMR and ESI-MS analyses and show for the first time that the trisaccharide repeat units of ECACYC and ECAPG are assembled by a common biosynthetic pathway.

The 1.75 Å Crystal Structure of Acetyl-CoA Synthetase Bound to Adenosine-5‘-propylphosphate and Coenzyme A†

Biochemistry ◽  
2003 ◽  
Vol 42 (10) ◽  
pp. 2866-2873 ◽  
Author(s):  
Andrew M. Gulick ◽  
Vincent J. Starai ◽  
Alexander R. Horswill ◽  
Kristen M. Homick ◽  
Jorge C. Escalante-Semerena
Keyword(s):  
Crystal Structure ◽  
Coenzyme A ◽  
Acetyl Coa

scholarly journals Mutation in a Gene Required for Lipopolysaccharide and Enterobacterial Common Antigen Biosynthesis Affects Virulence in the Plant Pathogen Erwinia carotovora subsp. atroseptica

1999 ◽  
Vol 12 (6) ◽  
pp. 499-507 ◽  
Author(s):  
I. K. Toth ◽  
C. J. Thorpe ◽  
S. D. Bentley ◽  
V. Mulholland ◽  
L. J. Hyman ◽  
...  
Keyword(s):  
Surface Defects ◽  
Single Gene ◽  
Outer Membrane Proteins ◽  
Pectate Lyase ◽  
Erwinia Carotovora ◽  
Enteric Bacteria ◽  
Common Antigen ◽  
In Planta ◽  
Enterobacterial Common Antigen ◽  
Multiple Cell

Spontaneous bacteriophage-resistant mutants of the phytopathogen Erwinia carotovora subsp. atroseptica (Eca) SCRI1043 were isolated and, out of 40, two were found to exhibit reduced virulence in planta. One of these mutants, A5/22, showed multiple cell surface defects including alterations in synthesis of outer membrane proteins, lipopolysaccharide (LPS), enterobacterial common antigen (ECA), and flagella. Mutant A5/22 also showed reduced synthesis of the exoenzymes pectate lyase (Pel) and cellulase (Cel), major virulence factors for this pathogen. Genetic analysis revealed the pronounced pleiotropic mutant phenotype to be due to a defect in a single gene (rffG) that, in Escherichia coli, is involved in the production of ECA. We also show that while other enteric bacteria possess duplicate homologues of this gene dedicated separately to synthesis of LPS and ECA, Eca has a single gene.

Occurrence and structure of cyclic Enterobacterial Common Antigen in Escherichia coli O157:H−

2012 ◽  
Vol 363 ◽  
pp. 29-32 ◽  
Author(s):  
Eleonora Fregolino ◽  
Radka Ivanova ◽  
Rosa Lanzetta ◽  
Antonio Molinaro ◽  
Michelangelo Parrilli ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Escherichia Coli O157 ◽  
Common Antigen ◽  
Enterobacterial Common Antigen

scholarly journals Properties of Succinyl-Coenzyme A:l-Malate Coenzyme A Transferase and Its Role in the Autotrophic 3-Hydroxypropionate Cycle of Chloroflexus aurantiacus

2006 ◽  
Vol 188 (7) ◽  
pp. 2646-2655 ◽  
Author(s):  
Silke Friedmann ◽  
Astrid Steindorf ◽  
Birgit E. Alber ◽  
Georg Fuchs
Keyword(s):  
Escherichia Coli ◽  
Coenzyme A ◽  
Chloroflexus Aurantiacus ◽  
Class Iii ◽  
Terminal Amino Acid ◽  
Acetyl Coa ◽  
Acetyl Coenzyme A ◽  
Coa Transferase ◽  
Terminal Amino ◽  
Terminal Amino Acid Sequence

ABSTRACT The 3-hydroxypropionate cycle has been proposed to operate as the autotrophic CO2 fixation pathway in the phototrophic bacterium Chloroflexus aurantiacus. In this pathway, acetyl coenzyme A (acetyl-CoA) and two bicarbonate molecules are converted to malate. Acetyl-CoA is regenerated from malyl-CoA by l-malyl-CoA lyase. The enzyme forming malyl-CoA, succinyl-CoA:l-malate coenzyme A transferase, was purified. Based on the N-terminal amino acid sequence of its two subunits, the corresponding genes were identified on a gene cluster which also contains the gene for l-malyl-CoA lyase, the subsequent enzyme in the pathway. Both enzymes were severalfold up-regulated under autotrophic conditions, which is in line with their proposed function in CO2 fixation. The two CoA transferase genes were cloned and heterologously expressed in Escherichia coli, and the recombinant enzyme was purified and studied. Succinyl-CoA:l-malate CoA transferase forms a large (αβ)n complex consisting of 46- and 44-kDa subunits and catalyzes the reversible reaction succinyl-CoA + l-malate → succinate + l-malyl-CoA. It is specific for succinyl-CoA as the CoA donor but accepts l-citramalate instead of l-malate as the CoA acceptor; the corresponding d-stereoisomers are not accepted. The enzyme is a member of the class III of the CoA transferase family. The demonstration of the missing CoA transferase closes the last gap in the proposed 3-hydroxypropionate cycle.

scholarly journals Mutations in enterobacterial common antigen biosynthesis restore outer membrane barrier function in Escherichia coli tol-pal mutants

2018 ◽  
Author(s):  
Xiang’Er Jiang ◽  
Wee Boon Tan ◽  
Rahul Shrivastava ◽  
Deborah Chwee San Seow ◽  
Swaine Lin Chen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Barrier Function ◽  
Lipid Homeostasis ◽  
Common Antigen ◽  
Potential Mechanism ◽  
External Threats ◽  
Enterobacterial Common Antigen ◽  
Lipid Species ◽  
Membrane Barrier

SummaryThe outer membrane (OM) is an essential component of the Gram-negative bacterial envelope that protects cells against external threats. To maintain a functional OM, cells require distinct mechanisms to ensure balance of proteins and lipids in the membrane. Mutations in OM biogenesis and/or homeostasis pathways often result in permeability defects, but how molecular changes in the OM affect barrier function is unclear. Here, we seek potential mechanism(s) that can alleviate permeability defects in Escherichia coli cells lacking the Tol-Pal complex, which accumulate excess PLs in the OM. We identify mutations in enterobacterial common antigen (ECA) biosynthesis that re-establish OM barrier function against large hydrophilic molecules, yet did not restore lipid homeostasis. Furthermore, we demonstrate that build-up of biosynthetic intermediates, but not loss of ECA itself, contributes to the rescue. This suppression of OM phenotypes is unrelated to known effects that accumulation of ECA intermediates have on the cell wall. Finally, we reveal that an unusual diacylglycerol pyrophosphoryl-linked lipid species also accumulates in ECA mutants, and might play a role in the rescue phenotype. Our work provides insights into how OM barrier function can be restored independent of lipid homeostasis, and highlights previously unappreciated effects of ECA-related species in OM biology.

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