The X-ray crystallographic structure of Escherichia coli branching enzyme

Branching enzyme catalyzes the formation of alpha-1,6 branch points in either glycogen or starch. We report the 2.3-Angstrom crystal structure of glycogen branching enzyme from Escherichia coli. The enzyme consists of three major domains, an NH2-terminal seven-stranded beta-sandwich domain, a COOH-terminal domain, and a central alpha/beta-barrel domain containing the enzyme active site. While the central domain is similar to that of all the other amylase family enzymes, branching enzyme shares the structure of all three domains only with isoamylase. Oligosaccharide binding was modeled or branching enzyme using the enzyme-oligosaccharide complex structures of various alpha-amylases and cyclodextrin glucanotransferase and residues were implicated in oligosaccharide binding. While most of the oligosaccharides modeled well in the branching enzyme structure, an approximate 50degrees rotation between two of the glucose units was required to avoid steric clashes with Trp(298) of branching enzyme. A similar rotation was observed in the mammalian alpha-amylase structure caused by an equivalent tryptophan residue in this structure. It appears that there are two binding modes for oligosaccharides in these structures depending on the identity and location of this aromatic residue.

FUNCTIONAL ALPHA-TROPOMYOSIN PRODUCED IN ESCHERICHIA-COLI - A DIPEPTIDE EXTENSION CAN SUBSTITUTE THE AMINO-TERMINAL ACETYL GROUP

Unlike the muscle protein, alpha-tropomyosin expressed in Escherichia coli does not bind actin, does not exhibit head-to-tail polymerization, and does not inhibit actomyosin ATPase activity in the absence of troponin. The only chemical difference between recombinant and muscle tropomyosins is that the first methionine is not acetylated in the recombinant protein (Hitchcock-DeGregori, S. E., and Heald, R. W. (1987) J. Biol. Chem. 262, 9730-9735). We expressed three fusion tropomyosins in E. coli with 2, 3, and 17 amino acids fused to its amino terminus. Ah three fusions restored actin binding, head-to-tail polymerization, and the capacity to inhibit the actomyosin ATPase to these unacetylated tropomyosins. Unlike larger fusions, the small fusions of 2 and 3 amino acids do not interfere with regulatory function. Therefore the presence of a fused dipeptide at the amino terminus of unacetylated tropomyosin is sufficient to replace the function of the N-acetyl group present in muscle tropomyosin. A structural interpretation for the function of the acetyl group, based on our results and the coiled coil structure of tropomyosin, is presented.

Structure and catalytic mechanism of glucosamine 6-phosphate deaminase from Escherichia coli at 2.1 Å resolution

Background: Glucosamine 6-phosphate deaminase from Escherichia coli is an allosteric hexameric enzyme which catalyzes the reversible conversion of D-glucosamine 6-phosphate into D-fructose 6-phosphate and ammonium ion and is activated by N-acetyl-D-glucosamine 6-phosphate. Mechanistically, it belongs to the group of aldose-ketose isomerases, but its reaction also accomplishes a simultaneous amination/deamination. The determination of the structure of this protein provides fundamental knowledge for understanding its mode of action and the nature of allosteric conformational changes that regulate its function. Results: The crystal structure of glucosamine 6-phosphate deaminase with bound phosphate ions is presented at 2.1 Å resolution together with the refined structures of the enzyme in complexes with its allosteric activator and with a competitive inhibitor. The protein fold can be described as a modified NAD-binding domain. Conclusions: From the similarities between the three presented structures, it is concluded that these represent the enzymatically active R state conformer. A mechanism for the deaminase reaction is proposed. It comprises steps to open the pyranose ring of the substrate and a sequence of general base-catalyzed reactions to bring about isomerization and deamination, with Asp72 playing a key role as a proton exchanger.

Avaliação dos padrões de susceptibilidade antimicrobianas e sorogrupos de cepas de Escherichia coli isoladas de bovinos leiteiros, portadoras e não portadoras dos genes stx1, stx2 e eae

Pós-graduação em Microbiologia Agropecuária - FCAV

Diversidade genética, fatores de virulência e perfis de susceptibilidade a antimicrobianos de isolados de Escherichia coli provenientes do útero, da boca e das fezes de cadelas com piometra

Pós-graduação em Microbiologia Agropecuária - FCAV

Multidrug-resistant Shiga toxin-producing Escherichia coli in dogs with diarrhea

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Isolation of extraintestinal pathogenic Escherichia coli from diarrheic dogs and their antimicrobial resistance profile

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Shigatoxigenic Escherichia coli serogroups O157, O111 and O113 in feces, water and milk samples from dairy farms

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Characterization of Escherichia coli isolated from carcasses of beef cattle during their processing at an abattoir in Brazil

Beef carcass sponge samples collected between March 2003 and August 2005 at an abattoir in Brazil were surveyed for the presence of Shiga toxin-producing Escherichia coli (STEC). Only one carcass among the 80 tested showed a STEC, stx2-encoding gene by PCR amplification. The frequency of carcass contamination by E. coli during processing was tested at three situations, respectively: preevisceration, postevisceration and postprocessing, during the rain and dry seasons. The prevalence of E. coli at the three points was of 30.0%, 70.0%, 27.5% in the rain season and of 22.5%, 55.0%, 17.5% during the dry season, respectively. The E. coli isolates exhibited a high level (45.0%) of multidrug resistance to two or more antimicrobial agents. (c) 2006 Elsevier B.V. All rights reserved.

Inibição do crescimento de Escherichia coli isolada de Queijo Minas Frescal por Lactobacillus acidophilus

Escherichia coli faz parte da microbiota anaeróbica facultativa normal, sendo também considerada um dos maiores patógenos entéricos predominantes no cólon dos animais e homem. Neste trabalho, realizaram-se ensaios in vitro para avaliar o grau de atividade antagonista de cinco cepas de Lactobacillus acidophilus, com capacidade probiótica sobre Escherichia coli BIA 26 (STEC) isolada de queijo Minas Frescal. Para tanto, foi utilizado o teste de inibição através do método de dupla camada em triplicata para avaliar zonas de inibição de crescimento. Todas as cepas de Lactobacillus mostraram-se capazes de inibir a E. coli, com zonas de inibição variando de 12 a 15mm de diâmetro, sendo que a maioria apresentou 14mm de diâmetro, consideradas como fortes halos de inibição.

Phenotypical characterization and adhesin identification in Escherichia coli strains isolated from dogs with urinary tract infections

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Virulence factors of Escherichia coli isolated from diarrheic calves

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Virulence properties and antimicrobial susceptibility of Shiga toxin-producing Escherichia coli strains isolated from healthy cattle from Parana State, Brazil

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Virulence Genes in Isolates of Escherichia coli from Samples of Milk and Feces from Dairy Cattle

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)