Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:6.3.2.3 (glutathione synthetase)
678 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Gamma-glutamylcysteine synthetase (gamma-GCS) and glutathione synthetase (GS), distinct enzymes that together account for glutathione (GSH) synthesis, have been isolated and characterized from several Gram-negative prokaryotes and from numerous eukaryotes including mammals, amphibians, plants, yeast, and protozoa. Glutathione synthesis is relatively uncommon among the Gram-positive bacteria, and, to date, neither the genes nor the proteins involved have been identified. In the present report, we show that crude extracts of Streptococcus agalactiae catalyze the gamma-GCS and GS reactions and can synthesize GSH from its constituent amino acids. The putative gene for S. agalactiae gamma-GCS was identified and cloned, and the corresponding protein was expressed and purified. Surprisingly, it was found that the isolated enzyme catalyzes both the ATP-dependent synthesis of L-gamma-glutamyl-L-cysteine from L-glutamate and L-cysteine and the ATP-dependent synthesis of GSH from L-gamma-glutamyl-L-cysteine and glycine. This novel bifunctional enzyme, referred to as gamma-GCS-GS, has been characterized in terms of catalytic activity, substrate specificity, and inhibition by GSH, cystamine, and transition state analog sulfoximines. The N-terminal 518 amino acids of gamma-GCS-GS (total M(r) 85,000) show 32% identity and 43% similarity with E. coli gamma-GCS (M(r) 58,000), but the C-terminal putative GS domain (remaining 202 amino acids) of gamma-GCS-GS shows no significant homology with known GS sequences. The C terminus (360 amino acids) is, however, homologous to D-Ala, D-Ala ligase (24% identity; 38% similarity), an enzyme having the same protein fold as known GS proteins. These results are discussed in terms of the evolution of GSH synthesis and the possible occurrence of a similar bifunctional GSH synthesis enzyme in other bacterial species.
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PMID:Glutathione synthesis in Streptococcus agalactiae. One protein accounts for gamma-glutamylcysteine synthetase and glutathione synthetase activities. 1564 37

Glutathione (GSH) is in a constant state of metabolic turnover. Because it is actively synthesized, it also must be degraded. In the first step of GSH synthesis, an amide linkage is formed between cysteine and glutamate catalyzed by gamma-glutamylcysteine synthetase. GSH synthetase catalyzes the reaction between amine residue of glycine and the cysteine carboxyl from gamma-glutamylcysteine dipeptide to form GSH. GSH is transported out of the cell and degraded by the membrane-bound enzyme gammaGT, which removes the gamma-glutamyl moiety, and by dipeptidases, which remove the glycine moiety. Glutathione is present in most of the plants and animals' tissues that constitute human diet. Thiol redox cycles play central roles in the antioxidant defense network. Lipoate and vitamins and other reducing factors affect the increase in glutathione concentrations in cells by the rise of the concentrations of reduced cysteine. The level of GSH in humans may be increased by taking different glutathione monoester (drug) or factors reducing cystyne to cysteine and increasing availability of this amino acid to GSH synthesis. GSH plays a critical role in cellular mechanisms that lead to cell death. The cancer cells resistant to apoptosis have higher intracellular GSH levels. The fact that numerous diseases are induced by RFT (that cause glutathione depletion) it seems that an in-depth study of the dietetic and pharmacological manners of manipulation of the GSH amount and availability may become in future a tool of great importance in the prevention of many illnesses.
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PMID:[Glutathione: its biosynthesis, induction agents and concentrations in selected diseases]. 1588 20

Glutathione is the predominant low-molecular-weight peptide thiol present in living organisms and plays a key role in protecting cells against oxygen toxicity. Until now, glutathione synthesis was thought to occur solely through the consecutive action of two physically separate enzymes, gamma-glutamylcysteine ligase and glutathione synthetase. In this report we demonstrate that Listeria monocytogenes contains a novel multidomain protein (termed GshF) that carries out complete synthesis of glutathione. Evidence for this comes from experiments which showed that in vitro recombinant GshF directs the formation of glutathione from its constituent amino acids and the in vivo effect of a mutation in GshF that abolishes glutathione synthesis, results in accumulation of the intermediate gamma-glutamylcysteine, and causes hypersensitivity to oxidative agents. We identified GshF orthologs, consisting of a gamma-glutamylcysteine ligase (GshA) domain fused to an ATP-grasp domain, in 20 gram-positive and gram-negative bacteria. Remarkably, 95% of these bacteria are mammalian pathogens. A plausible origin for GshF-dependent glutathione biosynthesis in these bacteria was the recruitment by a GshA ancestor gene of an ATP-grasp gene and the subsequent spread of the fusion gene between mammalian hosts, most likely by horizontal gene transfer.
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PMID:A multidomain fusion protein in Listeria monocytogenes catalyzes the two primary activities for glutathione biosynthesis. 1590 9

Glutathione (GSH), one of the most important antioxidants in the eukaryotic organism, is synthesized in a two-step procedure where the last step is catalysed by the enzyme glutathione synthetase (GSS). GSS deficiency is inherited autosomal recessively, and patients with this disease can be divided into three groups, according to their clinical phenotype. Mildly affected patients have mutations affecting the stability of the enzyme, causing a compensated haemolytic anaemia; moderately affected patients have, in addition, metabolic acidosis; and severely affected patients also develop neurological defects and show increased susceptibility to bacterial infections. Moderately and severely affected patients have mutations that compromise the catalytic properties of the enzyme. 5-Oxoprolinuria appears in all three groups, but is more pronounced in the two latter groups. Today, no cure can be offered these patients; they are given vitamins C and E to boost their antioxidant levels, and bicarbonate to correct metabolic acidosis.
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PMID:Glutathione synthetase deficiency. 1599 Sep 54

Glutathione (gamma-glu-cys-gly; GSH) is usually present at high concentrations in most living cells, being the major reservoir of non-protein reduced sulfur. Because of its unique redox and nucleophilic properties, GSH serves in bio-reductive reactions as an important line of defense against reactive oxygen species, xenobiotics and heavy metals. GSH is synthesized from its constituent amino acids by two ATP-dependent reactions catalyzed by gamma-glutamylcysteine synthetase and glutathione synthetase. In yeast, these enzymes are found in the cytosol, whereas in plants they are located in the cytosol and chloroplast. In protists, their location is not well established. In turn, the sulfur assimilation pathway, which leads to cysteine biosynthesis, involves high and low affinity sulfate transporters, and the enzymes ATP sulfurylase, APS kinase, PAPS reductase or APS reductase, sulfite reductase, serine acetyl transferase, O-acetylserine/O-acetylhomoserine sulfhydrylase and, in some organisms, also cystathionine beta-synthase and cystathionine gamma-lyase. The biochemical and genetic regulation of these pathways is affected by oxidative stress, sulfur deficiency and heavy metal exposure. Cells cope with heavy metal stress using different mechanisms, such as complexation and compartmentation. One of these mechanisms in some yeast, plants and protists is the enhanced synthesis of the heavy metal-chelating molecules GSH and phytochelatins, which are formed from GSH by phytochelatin synthase (PCS) in a heavy metal-dependent reaction; Cd(2+) is the most potent activator of PCS. In this work, we review the biochemical and genetic mechanisms involved in the regulation of sulfate assimilation-reduction and GSH metabolism when yeast, plants and protists are challenged by Cd(2+).
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PMID:Sulfur assimilation and glutathione metabolism under cadmium stress in yeast, protists and plants. 1610 96

Glutathione (GSH) plays several roles in cell metabolism such as redox state regulation, oxidative stress control, and protection against xenobiotics and heavy metals. GSH is synthesized in two steps catalysed by gamma-glutamylcysteine synthetase (gamma-ECS) and glutathione synthetase. gamma-ECS is feedback inhibited by GSH, which has led to the proposal that this enzyme acts as the rate-limiting step in the pathway. Thus far, the study of GSH metabolism has been confined to GSH synthesis (GSH supply), without considering the GSH-consuming enzymes (GSH demand). Several works have shown that the demand block of enzymes may have a significant control on a pathway; therefore, we hypothesize that GSH-consuming enzymes may exert some control on GSH synthesis. A kinetic model of GSH and phytochelatin synthesis in plants was constructed using the software GEPASI and the kinetic data available in the literature. The main conclusions drawn by the model concerning metabolic control analysis are (1) gamma-ECS is indeed a rate-limiting step in GSH synthesis, but only if GSH-consuming enzymes are not taken into account. (2) At low demand, GSH-consuming enzymes exert significant flux-control on GSH synthesis whereas at high demand, supply and demand blocks share the control of flux. (3) In unstressed conditions, flux to GSH is controlled mainly by demand, so that gamma-ECS determines the degree of homeostasis of the GSH concentration. Under cadmium exposure, the GSH demand increases and flux-control is re-distributed almost equally between the supply and demand blocks. (4) To enhance phytochelatins synthesis without depleting the GSH pool, at least two enzymes (gamma-ECS and PCS) should be increased and/or, alternatively, a branching flux (GSH-S-transferases) could be partially diminished.
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PMID:Control of glutathione and phytochelatin synthesis under cadmium stress. Pathway modeling for plants. 1612 28

Glutathione (GSH) is synthesized by gamma-glutamylcysteine synthetase (gamma-GCS) and glutathione synthetase (GS) in living organisms. Recently, bifunctional fusion protein, termed gamma-GCS-GS catalyzing both gamma-GCS and GS reactions from gram-positive firmicutes Streptococcus agalactiae, has been reported. We revealed that in the gamma-GCS activity, S. agalactiae gamma-GCS-GS had different substrate specificities from those of Escherichia coli gamma-GCS. Furthermore, S. agalactiae gamma-GCS-GS synthesized several kinds of gamma-glutamyltripeptide, gamma-Glu-X(aa)-Gly, from free three amino acids. In Clostridium acetobutylicum, the genes encoding gamma-GCS and putative GS were found to be immediately adjacent by BLAST search, and had amino acid sequence homology with S. agalactiae gamma-GCS-GS, respectively. We confirmed that the proteins expressed from each gene showed gamma-GCS and GS activity, respectively. C. acetobutylicum GS had broad substrate specificities and synthesized several kinds of gamma-glutamyltripeptide, gamma-Glu-Cys-X(aa). Whereas the substrate specificities of gamma-GCS domain protein and GS domain protein of S. agalactiae gamma-GCS-GS were the same as those of S. agalactiae gamma-GCS-GS.
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PMID:Novel substrate specificity of glutathione synthesis enzymes from Streptococcus agalactiae and Clostridium acetobutylicum. 1712 67

Glutathione (GSH) is one of the main antioxidants in plants. Legumes have the specificity to produce a GSH homolog, homoglutathione (hGSH). We have investigated the regulation of GSH and hGSH synthesis by nitric oxide (NO) in Medicago truncatula roots. Analysis of the expression level of gamma-glutamylcysteine synthetase (gamma-ECS), glutathione synthetase (GSHS) and homoglutathione synthetase (hGSHS) after treatment with sodium nitroprusside (SNP) and nitrosoglutathione (GSNO), two NO-donors, showed that gamma-ecs and gshs genes are up regulated by NO treatment whereas hgshs expression is not. Differential accumulation of GSH was correlated to gene expression in SNP-treated roots. Our results provide the first evidence that GSH synthesis pathway is regulated by NO in plants and that there is a differential regulation between gshs and hgshs in M. truncatula.
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PMID:Glutathione synthesis is regulated by nitric oxide in Medicago truncatula roots. 1719 40

Glutathione is a tripeptide composed of glutamate, cysteine and glycine. Glutathione is present in millimolar concentrations in most mammalian cells and it is involved in several fundamental biological functions, including free radical scavenging, detoxification of xenobiotics and carcinogens, redox reactions, biosynthesis of DNA, proteins and leukotrienes, as well as neurotransmission/neuromodulation. Glutathione is metabolised via the gamma-glutamyl cycle, which is catalyzed by six enzymes. In man, hereditary deficiencies have been found in five of the six enzymes. Glutathione synthetase deficiency is the most frequently recognized disorder and, in its severe form, it is associated with hemolytic anemia, metabolic acidosis, 5-oxoprolinuria, central nervous system (CNS) damage and recurrent bacterial infections. Gamma-glutamylcysteine synthetase deficiency is also associated with hemolytic anemia, and some patients with this disorder show defects of neuromuscular function and generalized aminoaciduria. Gamma-glutamyl transpeptidase deficiency has been found in patients with CNS involvement and glutathionuria. 5-Oxoprolinase deficiency is associated with 5-oxoprolinuria but without a clear association with other symptoms. Dipeptidase deficiency has been described in one patient. All disorders are very rare and inherited in an autosomal recessive manner. Most of the mutations are leaky so that many patients have residual enzyme activity. Diagnosis is made by measuring the concentration of different metabolites in the gamma-glutamyl cycle, enzyme activity and in glutathione synthetase and gamma-glutamylcysteine synthetase deficiency, also by mutation analysis. Prenatal diagnosis has been preformed in glutathione synthetase deficiency. The prognosis is difficult to predict, as few patients are known, but seems to vary significantly between different patients. The aims of the treatment of glutathione synthesis defects are to avoid hemolytic crises and to increase the defense against reactive oxygen species. No treatment has been recommended for gamma-glutamyl transpeptidase, 5-oxoprolinase and dipeptidase deficiency.
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PMID:Inborn errors in the metabolism of glutathione. 1739 29

Glutathione is essential for maintaining the intracellular redox environment and is synthesized from gamma-glutamylcysteine, glycine, and ATP by glutathione synthetase (GS). To examine the reaction mechanism of a eukaryotic GS, 24 Arabidopsis thaliana GS (AtGS) mutants were kinetically characterized. Within the gamma-glutamylcysteine/glutathione-binding site, the S153A and S155A mutants displayed less than 4-fold changes in kinetic parameters with mutations of Glu-220 (E220A/E220Q), Gln-226 (Q226A/Q226N), and Arg-274 (R274A/R274K) at the distal end of the binding site resulting in 24-180-fold increases in the K(m) values for gamma-glutamylcysteine. Substitution of multiple residues interacting with ATP (K313M, K367M, and E429A/E429Q) or coordinating magnesium ions to ATP (E148A/E148Q, N150A/N150D, and E371A) yielded inactive protein because of compromised nucleotide binding, as determined by fluorescence titration. Other mutations in the ATP-binding site (E371Q, N376A, and K456M) resulted in greater than 30-fold decreases in affinity for ATP and up to 80-fold reductions in turnover rate. Mutation of Arg-132 and Arg-454, which are positioned at the interface of the two substrate-binding sites, affected the enzymatic activity differently. The R132A mutant was inactive, and the R132K mutant decreased k(cat) by 200-fold; however, both mutants bound ATP with K(d) values similar to wild-type enzyme. Minimal changes in kinetic parameters were observed with the R454K mutant, but the R454A mutant displayed a 160-fold decrease in k(cat). In addition, the R132K, R454A, and R454K mutations elevated the K(m) value for glycine up to 11-fold. Comparison of the pH profiles and the solvent deuterium isotope effects of A. thaliana GS and the Arg-132 and Arg-454 mutants also suggest distinct mechanistic roles for these residues. Based on these results, a catalytic mechanism for the eukaryotic GS is proposed.
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PMID:Reaction mechanism of glutathione synthetase from Arabidopsis thaliana: site-directed mutagenesis of active site residues. 1745 39


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