Redox-dependent stability of the γ-glutamylcysteine synthetase enzyme of Escherichia coli: a novel means of redox regulation

2013 ◽  
Vol 449 (3) ◽  
pp. 783-794 ◽  
Author(s):  
Shailesh Kumar ◽  
Neha Kasturia ◽  
Amit Sharma ◽  
Manish Datt ◽  
Anand K. Bachhawat
Keyword(s):  
Escherichia Coli ◽  
Disulfide Bond ◽  
Redox Regulation ◽  
Catalytic Efficiency ◽  
Reduced Form ◽  
Cysteine Residues ◽  
Glutathione Biosynthesis ◽  
Glutamylcysteine Synthetase ◽  
The Stability

Glutathione is a thiol-containing tripeptide that plays important roles in redox-related processes. The first step in glutathione biosynthesis is catalysed by γ-GCS (γ-glutamylcysteine synthetase). The crystal structure of Escherichia coli γ-GCS has revealed the presence of a disulfide bond. As the disulfide-bonding cysteine residues Cys372 and Cys395 are not well conserved among γ-GCS enzymes in this lineage, we have initiated a biochemical genetic strategy to investigate the functional importance of these and other cysteine residues. In a cysteine-free γ-GCS that was non-functional, suppressor analysis yielded combinations of cysteine and aromatic residues at the position of the disulfide bond, and one mutant that lacked any cysteine residues. Kinetic analysis of the wild-type and mutant enzymes revealed that the disulfide bond was not involved in determining the affinity of the enzyme towards its substrate, but had an important role in determining the stability of the protein, and its catalytic efficiency. We show that in vivo the γ-GCS enzyme can also exist in a reduced form and that the mutants lacking the disulfide bond show a decreased half-life. These results demonstrate a novel means of regulation of γ-GCS by the redox environment that works by an alteration in its stability.

In vivo formation of Cu,Zn superoxide dismutase disulfide bond in Escherichia coli

FEBS Letters ◽  
1999 ◽  
Vol 443 (3) ◽  
pp. 313-316 ◽  
Author(s):  
Andrea Battistoni ◽  
Anna Paola Mazzetti ◽  
Giuseppe Rotilio
Keyword(s):  
Escherichia Coli ◽  
Superoxide Dismutase ◽  
Disulfide Bond

scholarly journals In Vitro and In Vivo Assessment of the Potential of Escherichia coli Phages to Treat Infections and Survive Gastric Conditions

2021 ◽  
Vol 9 (9) ◽  
pp. 1869
Author(s):  
Joanna Kaczorowska ◽  
Eoghan Casey ◽  
Gabriele A. Lugli ◽  
Marco Ventura ◽  
David J. Clarke ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Galleria Mellonella ◽  
Enterotoxigenic Escherichia Coli ◽  
E Coli ◽  
Composite Mixture ◽  
Ph Conditions ◽  
The Stability ◽  
Higher Sensitivity

Enterotoxigenic Escherichia coli (ETEC) and Shigella ssp. infections are associated with high rates of mortality, especially in infants in developing countries. Due to increasing levels of global antibiotic resistance exhibited by many pathogenic organisms, alternative strategies to combat such infections are urgently required. In this study, we evaluated the stability of five coliphages (four Myoviridae and one Siphoviridae phage) over a range of pH conditions and in simulated gastric conditions. The Myoviridae phages were stable across the range of pH 2 to 7, while the Siphoviridae phage, JK16, exhibited higher sensitivity to low pH. A composite mixture of these five phages was tested in vivo in a Galleria mellonella model. The obtained data clearly shows potential in treating E. coli infections prophylactically.

scholarly journals The Lack of Mitochondrial Thioredoxin TRXo1 Affects In Vivo Alternative Oxidase Activity and Carbon Metabolism under Different Light Conditions

2019 ◽  
Vol 60 (11) ◽  
pp. 2369-2381 ◽  
Author(s):  
Igor Florez-Sarasa ◽  
Toshihiro Obata ◽  
N�stor Fern�ndez Del-Saz ◽  
Jean-Philippe Reichheld ◽  
Etienne H Meyer ◽  
...  
Keyword(s):  
Carbon Metabolism ◽  
Redox State ◽  
Redox Regulation ◽  
Alternative Oxidase ◽  
Tricarboxylic Acid Cycle ◽  
Reduced Form ◽  
Photosynthetic Parameters ◽  
Primary Metabolism ◽  
Redox Systems

Abstract The alternative oxidase (AOX) constitutes a nonphosphorylating pathway of electron transport in the mitochondrial respiratory chain that provides flexibility to energy and carbon primary metabolism. Its activity is regulated in vitro by the mitochondrial thioredoxin (TRX) system which reduces conserved cysteines residues of AOX. However, in vivo evidence for redox regulation of the AOX activity is still scarce. In the present study, the redox state, protein levels and in vivo activity of the AOX in parallel to photosynthetic parameters were determined in Arabidopsis knockout mutants lacking mitochondrial trxo1 under moderate (ML) and high light (HL) conditions, known to induce in vivo AOX activity. In addition, 13C- and 14C-labeling experiments together with metabolite profiling were performed to better understand the metabolic coordination between energy and carbon metabolism in the trxo1 mutants. Our results show that the in vivo AOX activity is higher in the trxo1 mutants at ML while the AOX redox state is apparently unaltered. These results suggest that mitochondrial thiol redox systems are responsible for maintaining AOX in its reduced form rather than regulating its activity in vivo. Moreover, the negative regulation of the tricarboxylic acid cycle by the TRX system is coordinated with the increased input of electrons into the AOX pathway. Under HL conditions, while AOX and photosynthesis displayed similar patterns in the mutants, photorespiration is restricted at the level of glycine decarboxylation most likely as a consequence of redox imbalance.

scholarly journals Role of the cysteine residues in Arabidopsis thaliana cyclophilin CYP20-3 in peptidyl-prolyl cis–trans isomerase and redox-related functions

2006 ◽  
Vol 401 (1) ◽  
pp. 287-297 ◽  
Author(s):  
Miriam Laxa ◽  
Janine König ◽  
Karl-Josef Dietz ◽  
Andrea Kandlbinder
Keyword(s):  
Conformational Changes ◽  
Protein Function ◽  
Redox Regulation ◽  
Catalytic Efficiency ◽  
Structural Rearrangement ◽  
Disulfide Bridge ◽  
Cysteine Residues ◽  
Active Centre ◽  
Independent Functions

Cyps (cyclophilins) are ubiquitous proteins of the immunophilin superfamily with proposed functions in protein folding, protein degradation, stress response and signal transduction. Conserved cysteine residues further suggest a role in redox regulation. In order to get insight into the conformational change mechanism and functional properties of the chloroplast-located CYP20-3, site-directed mutagenized cysteine→serine variants were generated and analysed for enzymatic and conformational properties under reducing and oxidizing conditions. Compared with the wild-type form, elimination of three out of the four cysteine residues decreased the catalytic efficiency of PPI (peptidyl-prolyl cis–trans isomerase) activity of the reduced CYP20-3, indicating a regulatory role of dithiol–disulfide transitions in protein function. Oxidation was accompanied by conformational changes with a predominant role in the structural rearrangement of the disulfide bridge formed between Cys54 and Cys171. The rather negative Em (midpoint redox potential) of −319 mV places CYP20-3 into the redox hierarchy of the chloroplast, suggesting the activation of CYP20-3 in the light under conditions of limited acceptor availability for photosynthesis as realized under environmental stress. Chloroplast Prx (peroxiredoxins) were identified as interacting partners of CYP20-3 in a DNA-protection assay. A catalytic role in the reduction of 2-Cys PrxA and 2-Cys PrxB was assigned to Cys129 and Cys171. In addition, it was shown that the isomerization and disulfide-reduction activities are two independent functions of CYP20-3 that both are regulated by the redox state of its active centre.

scholarly journals Activity of the Yap1 Transcription Factor in Saccharomyces cerevisiae Is Modulated by Methylglyoxal, a Metabolite Derived from Glycolysis

2004 ◽  
Vol 24 (19) ◽  
pp. 8753-8764 ◽  
Author(s):  
Kazuhiro Maeta ◽  
Shingo Izawa ◽  
Shoko Okazaki ◽  
Shusuke Kuge ◽  
Yoshiharu Inoue
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Factor ◽  
Disulfide Bond ◽  
Target Genes ◽  
Cysteine Residue ◽  
Cysteine Residues ◽  
Glyoxalase I ◽  
Toxic Metabolite ◽  
Basic Leucine Zipper

ABSTRACT Methylglyoxal (MG) is synthesized during glycolysis, although it inhibits cell growth in all types of organisms. Hence, it has long been asked why such a toxic metabolite is synthesized in vivo. Glyoxalase I is a major enzyme detoxifying MG. Here we show that the Yap1 transcription factor, which is critical for the oxidative-stress response in Saccharomyces cerevisiae, is constitutively concentrated in the nucleus and activates the expression of its target genes in a glyoxalase I-deficient mutant. Yap1 contains six cysteine residues in two cysteine-rich domains (CRDs), i.e., three cysteine residues clustering near the N terminus (n-CRD) and the remaining three cysteine residues near the C terminus (c-CRD). We reveal that any of the three cysteine residues in the c-CRD is sufficient for MG to allow Yap1 to translocate into the nucleus and to activate the expression of its target gene. A Yap1 mutant possessing only one cysteine residue in the c-CRD but no cysteine in the n-CRD and deletion of the basic leucine zipper domain can concentrate in the nucleus with MG treatment. However, substitution of all the cysteine residues in Yap1 abolishes the ability of this transcription factor to concentrate in the nucleus following MG treatment. The redox status of Yap1 is substantially unchanged, and protein(s) interaction with Yap1 through disulfide bond is hardly detected in cells treated with MG. Collectively, neither intermolecular nor intramolecular disulfide bond formation seems to be involved in Yap1 activation by MG. Moreover, we show that nucleocytoplasmic localization of Yap1 closely correlates with growth phase and intracellular MG level. We propose a novel regulatory pathway underlying Yap1 activation by a natural metabolite in the cell.

scholarly journals Binding of the Escherichia coli cyclic AMP receptor protein to DNA fragments containing consensus nucleotide sequences

1989 ◽  
Vol 261 (2) ◽  
pp. 649-653 ◽  
Author(s):  
K Gaston ◽  
A Kolb ◽  
S Busby
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Inverse Correlation ◽  
Nucleotide Sequences ◽  
Receptor Protein ◽  
Dna Fragments ◽  
Naturally Occurring ◽  
The Stability

Binding of the Escherichia coli CRP protein to DNA fragments carrying nucleotide sequences closely corresponding to the consensus is very tight with a dissociation time of over 2 h in our conditions. The concentration of cyclic AMP required for this binding is below the physiological range of intracellular cyclic AMP concentrations. Changes in nucleotide sequence at positions that are not well-conserved between different naturally-occurring CRP sites allow a more rapid dissociation of CRP-DNA complexes. There is an inverse correlation between the stability of CRP binding to sites in vitro and the repression by glucose of expression dependent on these sites in vivo: expression that is dependent on the tighter binding sites cannot be repressed by the inclusion of glucose in the growth medium.

scholarly journals (p)ppGpp Inhibits Polynucleotide Phosphorylase from Streptomyces but Not from Escherichia coli and Increases the Stability of Bulk mRNA in Streptomyces coelicolor

2010 ◽  
Vol 192 (17) ◽  
pp. 4275-4280 ◽  
Author(s):  
Marcha L. Gatewood ◽  
George H. Jones
Keyword(s):  
Escherichia Coli ◽  
Chemical Stability ◽  
Antibiotic Production ◽  
Streptomyces Coelicolor ◽  
Polynucleotide Phosphorylase ◽  
Streptomyces Antibioticus ◽  
E Coli ◽  
The Stability

ABSTRACT ppGpp regulates gene expression in a variety of bacteria and in plants. We proposed previously that ppGpp or its precursor, pppGpp [referred to collectively as (p)ppGpp], or both might regulate the activity of the enzyme polynucleotide phosphorylase in Streptomyces species. We have examined the effects of (p)ppGpp on the polymerization and phosphorolysis activities of PNPase from Streptomyces coelicolor, Streptomyces antibioticus, and Escherichia coli. We have shown that (p)ppGpp inhibits the activities of both Streptomyces PNPases but not the E. coli enzyme. The inhibition kinetics for polymerization using the Streptomyces enzymes are of the mixed noncompetitive type, suggesting that (p)ppGpp binds to a region other than the active site of the enzyme. ppGpp also inhibited the phosphorolysis of a model RNA substrate derived from the rpsO-pnp operon of S. coelicolor. We have shown further that the chemical stability of mRNA increases during the stationary phase in S. coelicolor and that induction of a plasmid-borne copy of relA in a relA-null mutant increases the chemical stability of bulk mRNA as well. We speculate that the observed inhibition in vitro may reflect a role of ppGpp in the regulation of antibiotic production in vivo.

scholarly journals Impact of Global Transcriptional Regulation by ArcA, ArcB, Cra, Crp, Cya, Fnr, and Mlc on Glucose Catabolism in Escherichia coli

2005 ◽  
Vol 187 (9) ◽  
pp. 3171-3179 ◽  
Author(s):  
Annik Perrenoud ◽  
Uwe Sauer
Keyword(s):  
Escherichia Coli ◽  
Transcriptional Regulation ◽  
Redox Regulation ◽  
Tca Cycle ◽  
Batch Cultures ◽  
E Coli ◽  
Glucose Catabolism ◽  
The Tca Cycle

ABSTRACT Even though transcriptional regulation plays a key role in establishing the metabolic network, the extent to which it actually controls the in vivo distribution of metabolic fluxes through different pathways is essentially unknown. Based on metabolism-wide quantification of intracellular fluxes, we systematically elucidated the relevance of global transcriptional regulation by ArcA, ArcB, Cra, Crp, Cya, Fnr, and Mlc for aerobic glucose catabolism in batch cultures of Escherichia coli. Knockouts of ArcB, Cra, Fnr, and Mlc were phenotypically silent, while deletion of the catabolite repression regulators Crp and Cya resulted in a pronounced slow-growth phenotype but had only a nonspecific effect on the actual flux distribution. Knockout of ArcA-dependent redox regulation, however, increased the aerobic tricarboxylic acid (TCA) cycle activity by over 60%. Like aerobic conditions, anaerobic derepression of TCA cycle enzymes in an ArcA mutant significantly increased the in vivo TCA flux when nitrate was present as an electron acceptor. The in vivo and in vitro data demonstrate that ArcA-dependent transcriptional regulation directly or indirectly controls TCA cycle flux in both aerobic and anaerobic glucose batch cultures of E. coli. This control goes well beyond the previously known ArcA-dependent regulation of the TCA cycle during microaerobiosis.

scholarly journals O2-evoked regulation of HIF-1α and NF-κB in perinatal lung epithelium requires glutathione biosynthesis

2000 ◽  
Vol 278 (3) ◽  
pp. L492-L503 ◽  
Author(s):  
John J. E. Haddad ◽  
Stephen C. Land
Keyword(s):  
Genetic Regulation ◽  
Hypoxia Inducible Factor ◽  
Late Gestation ◽  
Alveolar Type ◽  
Lung Epithelium ◽  
Binding Characteristics ◽  
Glutathione Biosynthesis ◽  
Glutamylcysteine Synthetase

To test the genetic capacity of the perinatal lung to respond to O2 shifts that coincide with the first respiratory movements, rat fetal alveolar type II (fATII) epithelial cells were cultured at fetal distal lung[Formula: see text] (23 Torr) and then exposed to postnatal (23 → 76 Torr; mild hyperoxic shift), moderate (23 → 152 Torr; moderate hyperoxic shift), or severe (23 → 722 Torr; severe hyperoxic shift) oxygenation. Nuclear abundance and consensus binding characteristics of hypoxia-inducible factor (HIF)-1α and nuclear factor (NF)-κB (Rel A/p65) plus glutathione biosynthetic capacity were determined. Maximal HIF-1α activation at 23 Torr was sustained over the postnatal shift in (Δ)[Formula: see text] and was elevated in vivo throughout late gestation. NF-κB was activated by the acute postnatal[Formula: see text] in fATII cells, becoming maximal with moderate and severe oxygenation in vitro and within 6 h of birth in vivo, declining thereafter. fATII cell and whole lung glutathione and GSH-to-GSSG ratio increased fourfold with a postnatal[Formula: see text] and were matched by threefold activity increases in γ-glutamylcysteine synthetase and glutathione synthase. GSH concentration depletion byl-buthionine-( S,R)-sulfoximine abrogated both HIF-1α and NF-κB activation, with HIF-1α showing a heightened sensitivity to GSH concentration. We conclude that O2-linked genetic regulation in perinatal lung epithelium is responsive to developmental changes in glutathione biosynthetic capacity.

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