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Escherichia coli produces polysaccharide capsules that, based on their mechanisms of synthesis and assembly, have been classified into four groups. The group 4 capsule (G4C) polysaccharide is frequently identical to that of the cognate lipopolysaccharide O side chain and has, therefore, also been termed the O-antigen capsule. The genes involved in the assembly of the group 1, 2, and 3 capsules have been described, but those required for G4C assembly remained obscure. We found that enteropathogenic E. coli (EPEC) produces G4C, and we identified an operon containing seven genes, ymcD, ymcC, ymcB, ymcA, yccZ, etp, and etk, which are required for formation of the capsule. The encoded proteins appear to constitute a polysaccharide secretion system. The G4C operon is absent from the genomes of enteroaggregative E. coli and uropathogenic E. coli. E. coil K-12 contains the G4C operon but does not express it, because of the presence of ISI at its promoter region. In contrast, EPEC, enterohemorrhagic E. coil, and Shigella species possess an intact G4C operon.

The amino-terminal domain of Yersinia pseudotuberculosis tyrosine phosphatase YopH, is monomeric in solution with the phosphopeptide binding site on a beta-hairpin structure. Data indicate that the peptide binding does not induce any long-range conformational changes in YopH.

The topology of the catalytic domain of Cdc25A, a cell cycle control phosphatase has been determined at a resolution of 2.3 angstrom units. The domain consists of an alpha/beta motif different from that of other phasphatases. However, it shares some similarity with a sulphur-transfer protein, rhodanese. The structural and functional roles of specific amino acid residues in this enzyme are presented.

Protein dephosphorylation is a chemical reaction involved in the regulation of physiological processes such as glycogen metabolism, signal transduction, and osmoregulation. There are two classes of enzymes that regulate protein dephosphorylation: protein kinases and protein phosphatases. The latter are further subdivided into two, metallo-protein phosphatases and protein tyrosine phosphatases. An overview of the structure and function of these enzymes is presented.

The tyrosine and dual-specificity phosphatases are involved in signaling, cell growth and differentiation, and the cell cycle. The enzymes share a common catalytic mechanism mediated by an active site cysteine, arginine and aspartic acid. Supplementary domains assist in targeting and substrate specificity.

Chemical modification and mutation experiments indicate the role played by Cys403, an invariant Cys residue in the formation of the phosphoenzyme intermediate during phosphoester hydrolysis. The active site motif of the Cys(X)5Arg forms a phosphate-binding loop, which is essential for catalytic activity of phosphatases. The Arg residue containing amino acids such as glycine, Ser & Thr is essential to maintain the structure of the binding loop.

Analysis of the structures of unliganded and tungstate-attached crystal forms of Yersinia protein tyrosine phosphatase (PTPases) reveals that the nucleophilic Cys-403, which forms a phosphocysteine intermediate during catalysis, is placed at the center of a unique phosphate-binding loop. This loop is present in the hydrogen bond sequences that stabilize a bound oxyanion and stimulate Cys-403 as a reactive thiolate. Tungstate binding initiates a conformational shift in PTPase that encloses the oxyanion and swings the vital catalytic residue Asp-356 by 6 A into the active region.

Most bacteria surround themselves with a cell wall, a strong meshwork consisting primarily of the polymerized aminosugar peptidoglycan (PG). PG is essential for structural maintenance of bacterial cells, and thus for viability. PG is also constantly synthesized and turned over; the latter process is mediated by PG cleavage enzymes, for example, the endopeptidases (EPs). EPs themselves are essential for growth but also promote lethal cell wall degradation after exposure to antibiotics that inhibit PG synthases (e.g., β-lactams). Thus, EPs are attractive targets for novel antibiotics and their adjuvants. However, we have a poor understanding of how these enzymes are regulated in vivo, depriving us of novel pathways for the development of such antibiotics. Here, we have solved crystal structures of the LysM/M23 family peptidase ShyA, the primary EP of the cholera pathogen Vibrio cholerae. Our data suggest that ShyA assumes two drastically different conformations: a more open form that allows for substrate binding and a closed form, which we predicted to be catalytically inactive. Mutations expected to promote the open conformation caused enhanced activity in vitro and in vivo, and these results were recapitulated in EPs from the divergent pathogens Neisseria gonorrheae and Escherichia coli. Our results suggest that LysM/M23 EPs are regulated via release of the inhibitory Domain 1 from the M23 active site, likely through conformational rearrangement in vivo. [ABSTRACT FROM AUTHOR]

YopH is a protein tyrosine phosphatase and an essential virulence determinant of the pathogenic bacteriumYersinia. Yersiniadelivers YopH into infected host cells using a type III secretion mechanism. YopH dephosphorylates several focal adhesion proteins including p130Cas in human epithelial cells, resulting in disruption of focal adhesions and cell detachment from the extracellular matrix. How the C-terminal protein tyrosine phosphatase domain of YopH targets specific substrates such as p130Cas in the complex milieu of the host cell has not been fully elucidated. An N-terminal non-catalytic domain of YopH binds p130Cas in a phosphotyrosine-dependent manner and functions as a novel substrate-targeting site. The structure of the YopH protein tyrosine phosphatase domain bound to a model phosphopeptide substrate was solved and the resulting structure revealed a second substrate-targeting site (‘site 2’) within the catalytic domain. Site 2 binds to p130Cas in a phosphotyrosine-dependent manner, and co-operates with the N-terminal domain (‘site 1’) to promote efficient recognition of p130Cas by YopH in epithelial cells. The identification of two substrate-targeting sites in YopH that co-operate to promote epithelial cell detachment and bacterial virulence reinforces the importance of protein–protein interactions for determining protein tyrosine phosphatase specificityin vivo, and highlights the sophisticated nature of microbial pathogenicity factors. [ABSTRACT FROM AUTHOR]