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)

The gene for the large subunit of glutathione synthetase (EC 6.3.2.3) of Schizosaccharomyces pombe was cloned from a S. pombe genomic DNA library by complementation of cadmium hypersensitivity of a glutathione synthetase deficient mutant of S. pombe. A long open reading frame was found in the cloned DNA sequence. Amino acid sequence predicted from the long open reading frame coincided with amino acid sequences of peptides obtained by V8 protease digestion of the large subunit of the purified glutathione synthetase. The glutathione synthetase deficient mutant which harbored plasmids containing the glutathione synthetase large subunit gene exhibited glutathione synthetase activity higher than the activity in the wild type strain, though the plasmid did not contain the gene for the small subunit of the enzyme.
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PMID:Cloning and sequencing of the gene encoding the large subunit of glutathione synthetase of Schizosaccharomyces pombe. 195 12

Schizosaccharomyces pombe synthesize small cadmium-binding peptides cadystin, structure of which is (gamma-Glu-Cys)n-Gly, in response to cadmium. Mutants unable to synthesize cadystin were found in the mutants hypersensitive to cadmium. Some of them lack activity of either gamma-glutamylcysteine synthetase (EC 6.3.2.2) or glutathione synthetase (EC 6.3.2.3), enzyme involved in glutathione biosynthesis. Some mutants have the same activity levels of these enzymes as wild type has. These results indicate that some steps of cadystin biosynthesis are catalyzed by the enzymes catalyzing glutathione biosynthesis.
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PMID:Isolation of mutants of Schizosaccharomyces pombe unable to synthesize cadystin, small cadmium-binding peptides. 289 29

Glutathione (L-gamma-glutamyl-L-cysteinylglycine, GSH) plays an important role in the protection of plants against various types of stress caused by reactive oxygen species, gazeous pollutants, heavy metals and xenobiotics. A cDNA fragment containing the entire coding unit for glutathione synthetase (GSH2) of Arabidopsis thaliana was cloned by complementation of the methylglyoxal sensitivity of a gsh2 mutant of the yeast Saccharomyces cerevisiae. The cDNA encodes a protein of 478 amino acids (deduced Mr: 53783), bearing clear sequence similarities to GSH2 products from frog embryos (Xenopus laevis), rat kidney (Rattus norvegicus) and from the fission yeast (Schizosaccharomyces pombe). A highly conserved glycine-rich domain close to the carboxy-terminus was found in the GSH2 product and appears to be typical for eukaryotic glutathione synthetases. The Mr is similar to those of soluble animal enzymes, suggesting that the Arabidopsis gene also codes for a cytosolic protein. Genomic DNA-blot analysis indicates the presence of a single GSH2 gene. The yeast gsh2 mutant becomes resistant to methylglyoxal and cadmium after transformation with the plasmid bearing the Arabidopsis GSH2 cDNA. Moreover, this increased resistance is correlated to the restoration of GSH content from below detectability in mutants to about 50% of the wild-type levels in transformed cells.
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PMID:Cloning of Arabidopsis thaliana glutathione synthetase (GSH2) by functional complementation of a yeast gsh2 mutant. 861 43

Glutathione is essential for protecting plants from a range of environmental stresses, including heavy metals where it acts as a precursor for the synthesis of phytochelatins. A 1658 bp cDNA clone for glutathione synthetase (gsh2) was isolated from Arabidopsis thaliana plants that were actively synthesizing glutathione upon exposure to cadmium. The sequence of the clone revealed a protein with an estimated molecular mass of 53858 Da that was very similar to the protein from higher eukaryotes, was less similar to the gene from the fission yeast, Schizosaccharomyces pombe, and shared only a small region of similarity with the Escherichia coli protein. A 4.3 kb SstI fragment containing the genomic clone for glutathione synthetase was also isolated and sequenced. A comparison of the cDNA and genomic sequences revealed that the gene was composed of twelve exons. When the Arabidopsis cDNA cloned in a special shuttle vector was expressed in a S. pombe mutant deficient in glutathione synthetase activity, the plant cDNA was able to complement the yeast mutation. Glutathione synthetase activity was measurable in wild-type yeast cells, below detectable levels in the gsh2- mutant, and restored to substantial levels by the expression of the Arabidopsis cDNA. The S. pombe mutant expressing the plant cDNA had near wild type levels of total cellular thiols, 109Cd2+ binding activity, and cadmium resistance. Since the Arabidopsis cDNA was under control of a thiamine-repressible promoter, growth of the transformed yeast on thiamine-free medium increased expression of the cDNA resulting in increases in cadmium resistance.
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PMID:Cloning of the cDNA and genomic clones for glutathione synthetase from Arabidopsis thaliana and complementation of a gsh2 mutant in fission yeast. 891 26

Glutathione synthetase predicted from the reported gene sequence from Schizosaccharomyces pombe is substantially smaller than the equivalent protein predicted from the cDNAs sequenced from Arabidopsis thaliana, Saccharomyces cerevisiae and other eukaryotes. Sequence alignments of the proteins encoded by the cDNA clones for glutathione synthetase from Arabidopsis and S. pombe show that the Arabidopsis protein contains 200 extra amino acids at the N-terminus. In order to test if this sequence is essential in the function of the protein, the full-length Arabidopsis protein and as two N-terminal deletions (Delta67-71 and Delta67-200) were expressed in S. pombe mutant MN101, which lacks endogenous glutathione synthetase activity. Although the wild-type plant cDNA could complement the yeast mutation, neither deletion mutant was able to restore glutathione-dependent cadmium resistance. When the three proteins were expressed as fusion proteins in Escherichia coli, they accumulated to the same level, but only the plasmid containing the full-length cDNA, pFLAG222, produced detectable enzyme activity in vitro. These results suggested that the N-terminus of the Arabidopsis glutathione synthetase is essential for its function and opened up the possibility that there was a sequencing error in the reported S. pombe sequence. Therefore the gsh2 sequence from wild-type S. pombe and the mutant strain MN101 were determined. The wild-type S. pombe gsh2 encodes a protein that is about the same length as that found in Arabidopsis, and the MN101 mutation involves a frameshift mutation early in the glutathione synthetase reading frame.
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PMID:Glutathione synthetase: similarities of the proteins from Schizosaccharomyces pombe and Arabidopsis thaliana. 929 Nov 32

Cadmium is a potent cell poison known to cause oxidative stress by increasing lipid peroxidation and/or by changing intracellular glutathione levels and to affect the ubiquitin/ATP-dependent proteolytic pathway. However, the cellular mechanisms involved in cadmium toxicity are still not well understood, especially in neuronal cells. To investigate the relationship between cadmium-induced oxidative stress and the ubiquitin/ATP-dependent pathway, we treated cultures of neuronal cells with different concentrations of the metal ion. In addition to decreases in glutathione levels, we observed marked increases in protein-mixed disulfides (Pr-SSGs) after exposure of HT4 cells (a mouse neuronal cell line) or rat primary mesencephalic cultures to Cd2+. The increases in intracellular levels of Pr-SSGs were concurrent with increases in the levels of ubiquitinated proteins (Ub proteins) when the HT4 cells were subjected to lower (25 microM or less) concentrations of cadmium. However, higher concentrations of cadmium (50 microM), which were toxic, led to increases in Pr-SSGs but inhibited ubiquitination, probably reflecting inhibition of ubiquitinating enzymes. The cadmium-induced changes in Pr-SSGs and Ub proteins were not affected when more than 85% of intracellular glutathione was removed from the cells by the glutathione synthetase inhibitor L-buthionine-(S,R)-sulfoximine. However, the reducing agent dithiothreitol, which prevented the build up of Pr-SSGs in the cell, also blocked the accumulation of Ub proteins induced by cadmium. In addition, dithiothreitol blocked the effects of the higher toxic (50 microM) concentrations of cadmium on cytotoxicity and on glutathione, Pr-SSGs, and Ub proteins. Together, these results strongly suggest that changes in the levels of intracellular Pr-SSGs and ubiquitin-protein conjugates in neuronal cells are responses closely associated with the disruption of intracellular sulfhydryl homeostasis caused by cadmium-mediated oxidative stress.
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PMID:Disruption of the intracellular sulfhydryl homeostasis by cadmium-induced oxidative stress leads to protein thiolation and ubiquitination in neuronal cells. 958 93

In roots of Brassica juncea L. cadmium (Cd) exposure (25 microM) induces a massive formation of phytochelatins (PCs), which is accompanied by an only moderate decrease (-20%) of the putative PC precursor glutathione (GSH). As PC formation in roots could be the result of local GSH de novo synthesis and/or depend on GSH import from the shoot, we have analyzed the expression of the enzymes involved in GSH synthesis in the root, namely OAS(thiol)lyase (OAS-TL; catalysing the last step in Cys biosynthesis), gamma-glutamylcysteine synthetase (gamma-ECS), and glutathione synthetase (GSHS). cDNA clones were isolated from a cDNA library prepared from heavy metal exposed roots. Protein sequences from cDNA clones encoding OAS-TL, gamma-ECS, and GSHS, all exhibited putative mitochondrial targeting sequences, however, for OAS-TL also two putative cytosolic isoforms were isolated. Furthermore, we have cloned several metallothionein cDNAs of the MT2 group. Northern blot analysis with coding region probes revealed that in roots of Cd-exposed plants transcript amounts for OAS-TL and GSHS were only moderately increased, whereas gamma-ECS mRNA showed a stronger increase. Expression analysis with 3'-UTR probes indicated that among the putative mitochondrial OAS-TL, gamma-ECS and GSHS isoforms only gamma-ECS was up-regulated in response to Cd exposure. Conversely, transcripts for MT2 appeared to be slightly reduced. The results indicate that in roots Cd-induced PC synthesis correlates with a moderate increase of expression of genes involved in GSH synthesis, the change for gamma-ECS being most pronounced.
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PMID:cDNA cloning and expression analysis of genes encoding GSH synthesis in roots of the heavy-metal accumulator Brassica juncea L.: evidence for Cd-induction of a putative mitochondrial gamma-glutamylcysteine synthetase isoform. 962 Feb 67

Glutathione plays a pivotal role in protecting plants from environmental stresses, oxidative stress, xenobiotics, and some heavy metals. Arabidopsis plants treated with cadmium or copper responded by increasing transcription of the genes for glutathione synthesis, gamma-glutamylcysteine synthetase and glutathione synthetase, as well as glutathione reductase. The response was specific for those metals whose toxicity is thought to be mitigated through phytochelatins, and other toxic and nontoxic metals did not alter mRNA levels. Feeding experiments suggested that neither oxidative stress, as results from exposure to H2O2, nor oxidized or reduced glutathione levels were responsible for activating transcription of these genes. Jasmonic acid also activated the same suite of genes, which suggests that it might be involved in the signal transduction pathway for copper and cadmium. Jasmonic acid treatment increased mRNA levels and the capacity for glutathione synthesis but did not alter the glutathione content in unstressed plants, which supports the idea that the glutathione concentration is controlled at multiple levels.
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PMID:Glutathione metabolic genes coordinately respond to heavy metals and jasmonic acid in Arabidopsis. 972 99

By complementation screening of a cadmium-sensitive Schizosaccharomyces pombe mutant deficient in phytochelatin synthesis, but with 44% of the wild-type glutathione content, we cloned a DNA fragment involved in phytochelatin synthesis. Sequence analysis revealed that it encodes the second enzyme involved in glutathione (GSH) biosynthesis, glutathione synthetase (GSH2) (E.C.6.3.2.3, Wang and Oliver, 1997). The mutant allele shows a single base-pair exchange at the 3' end of the reading frame leading to a single amino acid change from glycine to aspartate. This mutation leads to a significant reduction of phytochelatin synthesis, whereas glutathione synthesis is impaired to a far lesser extent. Complementation with the Arabidopsis thaliana GSH2 cDNA led to a partial restoration of phytochelatin synthesis. These data strongly suggest that the GSH2 gene encodes a bifunctional enzyme that is able to catalyse both the synthesis of GSH by adding glycine to the dipeptide (gammaGlu-Cys) and the synthesis of phytochelatins. The sequence has been submitted to EMBL, Accession No. Y08414.
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PMID:Biosynthesis of phytochelatins in the fission yeast. Phytochelatin synthesis: a second role for the glutathione synthetase gene of Schizosaccharomyces pombe. 1021 97

Two cell lines of tomato (Lycopersicon esculentum Mill cv VFNT-Cherry) were systematically compared for their capacity to tolerate cadmium. Unselected CdS cells died in the presence of 0.3 mM CdCl2. CdR6-0 cells, which were selected from CdS, survived and grew in medium supplemented with 0.3 mM CdCl2. Growth of CdR6-0 cells under this condition was accompanied by synthesis of cadmium-binding phytochelatins and maintenance of cellular glutathione (GSH) levels. CdR6-0 cells also exhibited increased tolerance to buthionine sulfoximine, in both the presence and absence of 0.1 mM CdCl2. The specific activity of [gamma]-glutamylcysteine synthetase (EC 6.3.2.2) was approximately 2-fold higher in CdR6-0 cells than in CdS cells, whereas there was no difference between cell lines in specific activity of GSH synthetase (EC 6.3.2.3). Increased activity of the first enzyme of GSH biosynthesis in CdR6-0 cells, presumably a result of selection for increased cadmium tolerance, provides an enhanced capacity to synthesize GSH and to maintain the production of phytochelatins in response to cadmium. This adaptation may contribute to the enhanced cadmium tolerance of CdR6-0 cells.
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PMID:Increased Activity of [gamma]-Glutamylcysteine Synthetase in Tomato Cells Selected for Cadmium Tolerance. 1223 24


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