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Chemistry of the mechanism-based inactivation of pyridoxal phosphate-dependent enzymes
No full textPh.D. in Biochemistry conseguito presso l'istituzione estera di livello universitario University of Wales, College of Cardiff. Titolo riconosciuto come equipollente al dottorato di ricerca in Biochimica italiano
Beyond acid resistance: identification of novel targets of the regulators of the gad system in Escherichia coli
No full textThe meeting is focused on microbial stresses, covering experimental approaches from detailed molecular dissection of individual components to understanding stress responses at the whole cell and the whole population levels. It will bring together researchers, both academic and industrial, from all over the world, with a shared interest in all aspects of microbial stresses. This year, the themes will cover both intracellular and environmental stresses, the links between stress responses and energy metabolism, and the impacts of stress on microbial productivity. A must for all microbial physiologists, geneticists, cell biologists, biochemists, and systems biologists!
Announced speakers:
Theme One: Intracellular and environmental stresses: how cells cope with them
Daniela De Biase, Michael Hecker, Frank Madeo, John Morrissey, Aurelio Serrano
Theme Two: Cell stress and energy metabolism
Eva-Mari Aro, Timothy J. Donohue, Vassily Hatzimanikatis, Luigi Palmieri
Theme Three: Cell stress and bio-based microbial productions
Eckhard Boles, Pascale Daran-Lapujade, Ryan Gill, Jens Nielsen
Scientific Committee
Paola Branduardi (chair), Dept of Biotechnology and Biosciences University of Milano-Bicocca (I)
Michael Sauer (co-chair), IAM University of Natural Resources and Life Sciences, Wien (A)
Peter Lund (co-chair), School of Biosciences, University of Birmingham (UK)
Jeff Cole, School of Biosciences, University of Birmingham (UK)
Eckhard Boles, Institut für Molekulare Biowissenschaften Goethe-Universität Frankfurt (D)
Toni Villaverde, Department of Genetics and Microbiology, Universitat Aut?noma de Barcelona (E)
John Morrissey, Microbiology Department Food Science Building University College Cork (Ireland)
Industrial Committee
Danilo Porro (Committee chair), Dept of Biotechnology and Biosciences University of Milano-Bicocca (I)
Jens Nielsen, Dept of Chemical and Biological Engineering, Chalmers University of Technology (S)
Laura Ruohonen, VTT Technical Research Centre of Finland, Espoo (F)
Gunnar Lidén, Department of Chemical Engineering Lund University (S)
Vassily Hatzimanikatis, Laboratory of Computational Systems Biotechnology (LCSB) Ecole Polytechnique Federale de Lausanne (CH)
Francesca De Ferra, Eni Refining & Marketing (I)
Satoshi Harashima, International Center for Biotechnology Osaka University (Japan)
Luis Oriani, Chemtex Italia, Mossi and Ghisolfi Group (I)Escherichia coli is by far the most studied prokaryote and an invaluable tool in the laboratory and for biotechnological applications. It is a typical member of the mammalian gut microbiota but it can also exist as six different “pathotypes” that cause serious illness in humans. The sequenced E. coli genomes, from commensal and pathogenic strains, exhibit a “conserved core” genome of approximately 2,200 genes (half of the genome) and a pangenome of over 12,000 genes. The acquisition by horizontal gene transfer of pathogenicity islands, virulence plasmids and other mobile genetic elements allows the genomes of pathogenic E. coli to reshape into new pathotypes. The severe outbreak caused by the E. coli strain of serotype O104:H4 in Germany in May-July 2011, with more than 4,000 cases in 13 European countries and over 50 deaths, is a recent, dramatic example.
Instrumental to host gut colonization is the ability of E. coli and other enteropathogenic bacteria to overcome the extremely acidic environment of the host stomach, the major bactericidal barrier of the gastrointestinal tract. In E. coli the glutamate-dependent acid resistance (GDAR) system plays a major role in protection form the extreme acid stress. Structural genes of this system are gadA, gadB and gadC, which encode two glutamate decarboxylase isoforms and a glutamate/γ-aminobutyrate (GABA) antiporter, respectively.
Crystallographic studies have elegantly shown that both GadA/B and GadC become active only when the intracellular pH falls below 5.6. Glutamate decarboxylation, with the concomitant consumption of H+, and Glu/GABA electrogenic antiport contribute to protecting the cells from the harmful intracellular levels of H+. Even though the gadA and gadBC genes are 2,1 Mb apart, their transcription is under the control of the same regulators, namely GadE, GadX, GadW, H-NS and RpoS. These regulators also affect the expression of 12 genes located in the acid fitness island (AFI).
In the complex regulatory network controlling the expression of the GDAR genes, GadX plays an important role, i.e. it activates the GDAR structural genes in stationary phase both directly and indirectly, via activation of gadE, the gene coding for the central GDAR regulator. In a previous work we identified the GadX binding site, a 42-bp sequence, in the regulatory regions of all the AFI genes and gadBC. More recently, by means of a genome wide approach (ChIP-chip) and in vitro and in vivo studies, we have obtained experimental evidence that GadX affects the expression of many more targets, approx. 100 genes, than previously expected. These are scattered along the genome. Some of them are known to play major roles in important physiological processes, only apparently unrelated to the response to acid stress
AIMS Microbiology
No full textAIMS Microbiology is an international Open Access journal devoted to publishing peer-reviewed, high quality, original papers in the field of microbiology. We publish the following article types: original research articles, reviews, editorials, letters, and conference reports
Proprietà strutturali, biochimiche e immunologiche della glutammato decarbossilasi (GAD) umana e di E. coli nella sua forma apoenzimatica.
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What makes E. coli glutamate decarboxylase an amazing object for basic and biotech research
No full textGlutamate decarboxylase (Gad; EC 4.1.1.15) is a pyridoxal 5’-phosphate (PLP)-dependent enzyme which decarboxylates glutamate with concomitant proton consumption, CO2 release and GABA production. In Escherichia coli there are two Gad isoforms, GadA and GadB, which share 99.3% sequence similarity and are biochemically undistinguishable. Both isoforms have an acidic pH optimum, are hexamers and are major structural components to the glutamate-dependent acid resistance system, which protects pathogenic and commensal strains of E. coli from the extreme acid stress encountered during transit through the host stomach (1).
In my laboratory and in collaboration with other groups (G. Capitani, PSI, Villigen Switzerland; M.C.R. Franssen, Wageningen University, The Netherlands) E. coli GadB is intensively studied. This enzyme undergoes spectroscopically detectable and strongly cooperative pH-dependent conformational change (2). The comparison of the enzyme’s crystal structures at pH 4.6 (active form) and 7.6 (inactive form) provided evidence that the pH change causes three major structural reorganizations (2). The first 15 residues of the N-terminal domain (residues 1-58) at acidic pH are required for recruitment of GadB to the membrane and for binding of chloride ions (2,3). The last 15 residues of the C-terminal small domain (residues 347-466) are ordered only at neutral pH, when they plug the active site funnel and form an entirely novel structure, an aldamine, with His465 side chain distal nitrogen making a reversible covalent bond with the PLP-Lys276 Schiff base (3). His465 has a massive influence on the equilibrium between active and inactive forms (4). Lastly, a β-hairpin (residue 300-313) in the large PLP-binding domain at neutral pH is required for fixing in place the C-terminal tail of a neighboring subunit (3), whereas at acidic pH provides residues that partecipate in catalysis.
E. coli GadB was also efficiently immobilised in calcium alginate beads (5). The industrial production of GABA from glutamic acid, which is abundant in waste streams from biofuel production, is regarded as an interesting alternative for the synthesis of nitrogen-containing bulk chemicals, thereby decreasing the dependency on fossil fuels (5). We designed and characterized GadB site specific mutants with a range of activity significantly extended toward alkaline pH, to be employed for the above applications
Glutamate-based acid resistance in Escherichia coli: biochemical and regulatory aspects
No full textEscherichia coli has the ability to resist severe acid stress, as that encountered during transit through the host stomach, and this is instrumental to host gut colonization. The glutamate-based acid resistance (AR) system plays a major role in the protection of the cell from the deleterious effects of a high-proton-concentration environment. Structural genes of this system are gadA, gadB and gadC, which encode two glutamate decarboxylase isoforms and a glutamate/γ-aminobutyrate (GABA) antiporter, respectively. Glutamate decarboxylation leads to both proton consumption and production of GABA, a neutral compound exported via the GadC antiporter. Even though the gadA and gadBC genes are 2,1 Mb apart, their transcription is under the control of the same regulators: GadE, GadX, GadW, H-NS and RpoS. These regulators also affect the expression of 12 genes located in the acid fitness island (AFI). We have identified the GadX (GadW) binding site, a 42 bp sequence, in the regulatory regions of gadA, gadBC, slp, hdeAB, gadE and gadY. All are AFI genes, but gadBC. In my talk I will show the most recent results from in vivo and in vitro analyses aimed at fully characterizing the GadX regulon. In addition I will show biochemical data on the decarboxylase’s intracellular activation/inactivation process and on additional effectors involved in GABA export
`Some like it acid': how Escherichia coli glutamate decarboxylase controls its intracellular activity in response to acid stress
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