scholarly journals Comparative Analysis of the Escherichia coli Ketopantoate Hydroxymethyltransferase Crystal Structure Confirms that It Is a Member of the (βα)8 Phosphoenolpyruvate/Pyruvate Superfamily

2003 ◽  
Vol 185 (14) ◽  
pp. 4163-4171 ◽  
Author(s):  
Florian Schmitzberger ◽  
Alison G. Smith ◽  
Chris Abell ◽  
Tom L. Blundell
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Comparative Analysis ◽  
Structural Analysis ◽  
Active Sites ◽  
Vitamin B ◽  
Biosynthesis Pathway ◽  
Hydroxymethyl Group ◽  
E Coli ◽  
Structural Homologues

ABSTRACT Escherichia coli ketopantoate hydroxymethyltransferase (KPHMT) catalyzes the first step in the biosynthesis pathway of pantothenate (vitamin B5), the transfer of a hydroxymethyl group onto α-ketoisovalerate. Here we describe a detailed comparative analysis of the KPHMT crystal structure and the identification of structural homologues, some of which have remarkable similarities in their active sites, modes of binding to substrates, and mechanisms. We show that KPHMT forms a family within the phosphoenolpyruvate/pyruvate superfamily. Based on the analysis, we propose that in this superfamily there should be a subdivision into two groups. This paper completes our structural analysis of the E. coli enzymes in the pantothenate pathway.

scholarly journals Threonine 57 is required for the post-translational activation ofEscherichia coliaspartate α-decarboxylase

2014 ◽  
Vol 70 (4) ◽  
pp. 1166-1172 ◽  
Author(s):  
Michael E. Webb ◽  
Briony A. Yorke ◽  
Tom Kershaw ◽  
Sarah Lovelock ◽  
Carina M. C. Lobley ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Intramolecular Rearrangement ◽  
Directed Mutagenesis ◽  
Vitamin B ◽  
Biosynthesis Pathway ◽  
Serine Residue ◽  
Translational Activation

Aspartate α-decarboxylase is a pyruvoyl-dependent decarboxylase required for the production of β-alanine in the bacterial pantothenate (vitamin B5) biosynthesis pathway. The pyruvoyl group is formedviathe intramolecular rearrangement of a serine residue to generate a backbone ester intermediate which is cleaved to generate an N-terminal pyruvoyl group. Site-directed mutagenesis of residues adjacent to the active site, including Tyr22, Thr57 and Tyr58, reveals that only mutation of Thr57 leads to changes in the degree of post-translational activation. The crystal structure of the site-directed mutant T57V is consistent with a non-rearranged backbone, supporting the hypothesis that Thr57 is required for the formation of the ester intermediate in activation.

Interaction specificity between the chaperone and proteolytic components of the cyanobacterial Clp protease

2012 ◽  
Vol 446 (2) ◽  
pp. 311-320 ◽  
Author(s):  
Anders Tryggvesson ◽  
Frida M. Ståhlberg ◽  
Axel Mogk ◽  
Kornelius Zeth ◽  
Adrian K. Clarke
Keyword(s):  
Escherichia Coli ◽  
Active Sites ◽  
Terminal Sequence ◽  
Core Complex ◽  
Clp Protease ◽  
E Coli ◽  
Loop Region ◽  
N Terminus ◽  
Specific Association ◽  
The Stability

The Clp protease is conserved among eubacteria and most eukaryotes, and uses ATP to drive protein substrate unfolding and translocation into a chamber of sequestered proteolytic active sites. In plant chloroplasts and cyanobacteria, the essential constitutive Clp protease consists of the Hsp100/ClpC chaperone partnering a proteolytic core of catalytic ClpP and noncatalytic ClpR subunits. In the present study, we have examined putative determinants conferring the highly specific association between ClpC and the ClpP3/R core from the model cyanobacterium Synechococcus elongatus. Two conserved sequences in the N-terminus of ClpR (tyrosine and proline motifs) and one in the N-terminus of ClpP3 (MPIG motif) were identified as being crucial for the ClpC–ClpP3/R association. These N-terminal domains also influence the stability of the ClpP3/R core complex itself. A unique C-terminal sequence was also found in plant and cyanobacterial ClpC orthologues just downstream of the P-loop region previously shown in Escherichia coli to be important for Hsp100 association to ClpP. This R motif in Synechococcus ClpC confers specificity for the ClpP3/R core and prevents association with E. coli ClpP; its removal from ClpC reverses this core specificity.

scholarly journals Whole-Genome Sequence of Phage-Resistant Strain Escherichia coli DH5α

2018 ◽  
Vol 6 (10) ◽  
Author(s):  
Jingchao Chen ◽  
Yi Li ◽  
Kun Zhang ◽  
Hailei Wang
Keyword(s):  
Escherichia Coli ◽  
Comparative Analysis ◽  
Genome Sequence ◽  
Resistant Strain ◽  
Resistance Mechanism ◽  
Whole Genome Sequence ◽  
Whole Genome ◽  
Content Type ◽  
E Coli ◽  
T4 Bacteriophage

ABSTRACT The genomes of many strains of Escherichia coli have been sequenced, as this organism is a classic model bacterium. Here, we report the genome sequence of Escherichia coli DH5α, which is resistant to a T4 bacteriophage (CCTCC AB 2015375), while its other homologous E. coli strains, such as E. coli BL21, DH10B, and MG1655, are not resistant to phage invasions. Thus, understanding of the genome of the DH5α strain, along with comparative analysis of its genome sequence along with other sequences of E. coli strains, may help to reveal the bacteriophage resistance mechanism of E. coli .

scholarly journals Initial Steps of Colicin E1 Import across the Outer Membrane of Escherichia coli

2007 ◽  
Vol 189 (7) ◽  
pp. 2667-2676 ◽  
Author(s):  
Muriel Masi ◽  
Phu Vuong ◽  
Matthew Humbard ◽  
Karen Malone ◽  
Rajeev Misra
Keyword(s):  
Escherichia Coli ◽  
Cell Surface ◽  
Outer Membrane ◽  
Vitamin B ◽  
Whole Cells ◽  
E Coli ◽  
Colicin E1 ◽  
Unfolded State ◽  
Translocation Domain

ABSTRACT Data suggest a two-receptor model for colicin E1 (ColE1) translocation across the outer membrane of Escherichia coli. ColE1 initially binds to the vitamin B12 receptor BtuB and then translocates through the TolC channel-tunnel, presumably in a mostly unfolded state. Here, we studied the early events in the import of ColE1. Using in vivo approaches, we show that ColE1 is cleaved when added to whole cells. This cleavage requires the presence of the receptor BtuB and the protease OmpT, but not that of TolC. Strains expressing OmpT cleaved ColE1 at K84 and K95 in the N-terminal translocation domain, leading to the removal of the TolQA box, which is essential for ColE1's cytotoxicity. Supported by additional in vivo data, this suggests that a function of OmpT is to degrade colicin at the cell surface and thus protect sensitive E. coli cells from infection by E colicins. A genetic strategy for isolating tolC mutations that confer resistance to ColE1, without affecting other TolC functions, is also described. We provide further in vivo evidence of the multistep interaction between TolC and ColE1 by using cross-linking followed by copurification via histidine-tagged TolC. First, secondary binding of ColE1 to TolC is dependent on primary binding to BtuB. Second, alterations to a residue in the TolC channel interfere with the translocation of ColE1 across the TolC pore rather than with the binding of ColE1 to TolC. In contrast, a substitution at a residue exposed on the cell surface abolishes both binding and translocation of ColE1.

scholarly journals Biosynthesis of heparin. Use of Escherichia coli K5 capsular polysaccharide as a model substrate in enzymic polymer-modification reactions

1991 ◽  
Vol 275 (1) ◽  
pp. 151-158 ◽  
Author(s):  
M Kusche ◽  
H H Hannesson ◽  
U Lindahl
Keyword(s):  
Escherichia Coli ◽  
Structural Analysis ◽  
Binding Sites ◽  
Proteinase Inhibitor ◽  
Capsular Polysaccharide ◽  
Polymer Modification ◽  
Sulphated Polysaccharide ◽  
E Coli ◽  
High Affinity ◽  
K5 Polysaccharide

A capsular polysaccharide from Escherichia coli K5 was previously found to have the same structure, [-(4)beta GlcA(1)→(4)alpha GlcNAc(1)-]n, as that of the non-sulphated precursor polysaccharide in heparin biosynthesis [Vann, Schmidt, Jann & Jann (1981) Eur. J. Biochem. 116, 359-364]. The K5 polysaccharide was N-deacetylated (by hydrazinolysis) and N-sulphated, and was then incubated with detergent-solubilized enzymes from a heparin-producing mouse mastocytoma, in the presence of adenosine 3′-phosphate 5′-phospho[35S] sulphate ([35S]PAPS). Structural analysis of the resulting 35S-labelled polysaccharide revealed the formation of all the major disaccharide units found in heparin. The identification of 2-O-[35S]sulphated IdoA (L-iduronic acid) as well as 6-O-[35S]sulphated GlcNSO3 units demonstrated that the modified K5 polysaccharide served as a substrate in the hexuronosyl C-5-epimerase and the major O-sulphotransferase reactions involved in the biosynthesis of heparin. The GlcA units of the native (N-acetylated) E. coli polysaccharide were attacked by the epimerase only when PAPS was present in the incubations, whereas those of the chemically N-sulphated polysaccharide were epimerized also in the absence of PAPS, in accord with the notion that N-sulphate groups are required for epimerization. With increasing concentrations of PAPS, the mono-O-sulphated disaccharide unit-IdoA(2-OSO3)-GlcNSO3- was progressively converted into the di-O-sulphated species -IdoA(2-OSO3)-GlcNSO3(6-OSO3)-. A small proportion of the 35S-labelled polysaccharide was found to bind with high affinity to the proteinase inhibitor antithrombin. This proportion increased with increasing concentration of PAPS up to a level corresponding to approximately 1-2% of the total incorporated 35S. The solubilized enzymes thus catalysed all the reactions required for the generation of functional antithrombin-binding sites.

scholarly journals Clonal study of avian Escherichia coli strains by fliC conserved-DNA-sequence regions analysis

2008 ◽  
Vol 28 (10) ◽  
pp. 508-514 ◽  
Author(s):  
Tatiana Amabile de Campos ◽  
Gerson Nakazato ◽  
Eliana Guedes Stehling ◽  
Marcelo Brocchi ◽  
Wanderley Dias da Silveira
Keyword(s):  
Escherichia Coli ◽  
Comparative Analysis ◽  
Dna Sequencing ◽  
Dna Sequence ◽  
Yersinia Enterocolitica ◽  
Proteus Mirabilis ◽  
Great Similarity ◽  
Flic Gene ◽  
E Coli ◽  
Zoonotic Risk

The clonal relationship among avian Escherichia coli strains and their genetic proximity with human pathogenic E. coli, Salmonela enterica, Yersinia enterocolitica and Proteus mirabilis, was determined by the DNA sequencing of the conserved 5' and 3'regions fliC gene (flagellin encoded gene). Among 30 commensal avian E. coli strains and 49 pathogenic avian E. coli strains (APEC), 24 commensal and 39 APEC strains harbored fliC gene with fragments size varying from 670bp to 1,900bp. The comparative analysis of these regions allowed the construction of a dendrogram of similarity possessing two main clusters: one compounded mainly by APEC strains and by H-antigens from human E. coli, and another one compounded by commensal avian E. coli strains, S. enterica, and by other H-antigens from human E. coli. Overall, this work demonstrated that fliC conserved regions may be associated with pathogenic clones of APEC strains, and also shows a great similarity among APEC and H-antigens of E. coli strains isolated from humans. These data, can add evidence that APEC strains can exhibit a zoonotic risk.

ELECTROSTATIC PROPERTIES OF PROMOTER RECOGNIZED BYE. COLIRNA POLYMERASE Eσ70

2006 ◽  
Vol 04 (02) ◽  
pp. 455-467 ◽  
Author(s):  
ANATOLY A. SOROKIN ◽  
ALEXANDR A. OSYPOV ◽  
TIMUR R. DZHELYADIN ◽  
PETR M. BESKARAVAINY ◽  
SVETLANA G. KAMZOLOVA
Keyword(s):  
Escherichia Coli ◽  
Comparative Analysis ◽  
Rna Polymerase ◽  
Α Subunit ◽  
Promoter Sequences ◽  
E Coli ◽  
Adp Ribosylation ◽  
Electrostatic Properties ◽  
Differential Recognition ◽  
T4 Phage

A comparative analysis of electrostatic patterns for 359 σ70-specific promoters and 359 nonpromoter regions on electrostatic map of Escherichia coli genome was carried out. It was found that DNA is not a uniformly charged molecule. There are some local inhomogeneities in its electrostatic profile which correlate with promoter sequences. Electrostatic patterns of promoter DNAs can be specified due to the presence of some distinctive motifs which differ for different promoter groups and may be involved as signal elements in differential recognition of various promoters by the enzyme. Some specific electrostatic elements which are responsible for modulating promoter activities due to ADP-ribosylation of RNA polymerase α-subunit were found in far upstream regions of T4 phage early promoters and E. coli ribosomal promoters.

scholarly journals Comparative Analysis of Attachment of Shiga-Toxigenic Escherichia coli and Salmonella Strains to Cultured HT-29 and Caco-2 Cell Lines

2009 ◽  
Vol 75 (6) ◽  
pp. 1796-1799 ◽  
Author(s):  
Glen E. Mellor ◽  
Rebecca M. Goulter ◽  
T. W. Raymond Chia ◽  
Gary A. Dykes
Keyword(s):  
Escherichia Coli ◽  
Comparative Analysis ◽  
Cell Line ◽  
Cell Lines ◽  
Cell Monolayers ◽  
E Coli ◽  
Ht 29

ABSTRACT The ability of Escherichia coli and Salmonella isolates to attach to Caco-2 and HT-29 cell monolayers was measured. All isolates displayed a greater ability to attach to Caco-2 cells than HT-29 cells, and overall E. coli isolates attached better to both cell lines than Salmonella isolates. Bacteria that were considered to be pathogenic displayed no greater ability to attach to cell lines than those that were not considered to be pathogenic. Additionally, no correlation was found between cell line attachment and previously determined hydrophobicity results.

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