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)

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.
Biochem J 1997 Sep 01
PMID:Glutathione synthetase: similarities of the proteins from Schizosaccharomyces pombe and Arabidopsis thaliana. 929 Nov 32

The gene homologous to glutathione synthetase of Escherichia coli was inactivated in the cyanobacterium Synechococcus sp. PCC 7942. The region of genomic DNA including the mutation site was isolated from the mutant by plasmid rescue and the native gene of the wild-type was cloned from a genomic DNA library of the wild-type using the flanking DNA as a probe. The wild-type gene, designated gshB, encodes a polypeptide of 323 amino acids with a molecular mass of 35 kDa. The deduced amino acid sequence resembles glutathione synthetases of bacteria, but not those of higher organisms. When gshB was overexpressed in E. coli, glutathione synthetase activity was increased markedly in the E. coli extract. In addition, the Synechococcus sp. PCC 7942 gshB mutants had lost their ability to synthesize glutathione. These findings demonstrate that the gshB gene of Synechococcus sp. PCC 7942 is a structural gene for glutathione synthetase and is involved in the biosynthesis of glutathione.
Microbiology (Reading) 1997 Sep
PMID:The gshB gene in the cyanobacterium Synechococcus sp. PCC 7942 encodes a functional glutathione synthetase. 930 72

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.
Plant Cell 1998 Sep
PMID:Glutathione metabolic genes coordinately respond to heavy metals and jasmonic acid in Arabidopsis. 972 99

We used mice deficient in gamma-glutamyl transpeptidase (GGT) to analyze the effects of GGT deficiency and altered thiol levels on gene expression in liver. GGT-deficient mice have markedly reduced levels of glutathione (GSH), cysteine, methionine, and cysteinylglycine in liver. Steady-state RNA levels of the catalytic subunit of gamma-glutamylcysteine synthetase (gamma-GCS), the rate-limiting enzyme in GSH synthesis, are elevated 4-fold in these mice, while those for glutathione synthetase (GSH syn) are elevated 2-fold. RNA levels of cystathionase (cystathionine gamma-lyase), a key enzyme in the synthesis of cysteine from methionine, are elevated approximately 3.5-fold. In contrast, levels of RNA coding for multidrug resistance protein 2 (MRP2), which transports GSH into bile, are half wild-type values. We found no change in RNA levels of enzymes related to oxidative injury (CuZn and Mn superoxide dismutases [SOD], catalase, and glutathione peroxidase). Similarly, RNA levels of glutathione reductase and ribonucleotide reductase were unchanged. Furthermore, in contrast to previous in vitro results, methyl methanesulfonate did not induce stress-activated signal transduction as measured by c-jun phosphorylation in livers of GGT-deficient mice, despite further depletion of GSH by buthionine sulfoximine. Our findings indicate that GGT deficiency itself and/or altered thiol levels regulate expression of genes involved in GSH metabolism, but have no effect on the expression of other antioxidant genes.
Hepatology 2000 Sep
PMID:Altered gene expression in the liver of gamma-glutamyl transpeptidase-deficient mice. 1096 Apr 49

Synthesis of GSH occurs via two enzymatic steps, the first is catalyzed by gamma-glutamylcysteine synthetase (GCS) and the second is catalyzed by GSH synthetase (GS). A heavy (HS) and light subunit (LS) make up GCS; regulation of both subunits have been well characterized, whereas regulation of GS is largely unknown. In this study, we examined the effects of treatments known to influence the gene expression of GCS subunits on GS expression. Insulin and hydrocortisone treatment of rat hepatocytes or ethanol-feeding of rats for 9 weeks, which increased the expression of GCS-HS only, had no influence on GS expression. However, two-thirds partial hepatectomy in rats which increased the expression of GCS-HS only, also increased GS expression. Treatment of hepatocytes or rats with diethyl maleate, buthionine sulfoximine, tert-butylhydroquinone, or thioacetamide, which increased the expression of both GCS subunits, increased the expression of GS. The GSH synthesis capacity increased 50-100% by treatments that increased only the GCS-HS expression, whereas it increased 161-200% by treatments that increased both GCS-HS and GS expression. Thioacetamide treatment of Chang cells increased cell GSH and GS expression by 50%, but had minimal influence on GCS subunits. Thus, GS induction can further increase the cell's GSH synthetic capacity and in some cells may be as important as GCS in determining the rate of GSH synthesis.
Biochim Biophys Acta 2000 Sep 07
PMID:Inducers of gamma-glutamylcysteine synthetase and their effects on glutathione synthetase expression. 1097 6

In Arabidopsis thaliana, trichome cells are specialized unicellular structures with uncertain functions. Based on earlier observations that one of the genes involved in cysteine biosynthesis (Atcys-3A) is highly expressed in trichomes, we have extended our studies in trichome cells to determine their capacity for glutathione (GSH) biosynthesis. First, we have analyzed by in situ hybridization the tissue-specific expression of the genes Atcys-3A and sat5, which encode O-acetylserine(thio)lyase (OASTL) and serine acetyltransferase (SAT), respectively, as well as gsh1 and gsh2, which encode gamma-glutamylcysteine synthetase and glutathione synthetase, respectively. The four genes are highly expressed in leaf trichomes of Arabidopsis, and their mRNA accumulate to high levels. Second, we have directly measured cytoplasmic GSH concentration in intact cells by laser-scanning microscopy after labeling with monochlorobimane as a GSH-specific probe. From these measurements, cytosolic GSH concentrations of 238+/-25, 80+/-2, and 144+/-19 microM were estimated for trichome, basement, and epidermal cells, respectively. Taking into account the volume of the cells measured using stereological techniques, the trichomes have a total GSH content more than 300-fold higher than the basement and epidermal cells. Third, after NaCl treatment, GSH biosynthesis is markedly decreased in trichomes. Atcys-3A, sat5, gsh1, and gsh2 mRNA levels show a decrease in transcript abundance, and [GSH](cyt) is reduced to 47+/-5 microM. These results suggest the important physiological significance of trichome cells related to GSH biosynthesis and their possible role as a sink during detoxification processes.
Proc Natl Acad Sci U S A 2000 Sep 26
PMID:Glutathione biosynthesis in Arabidopsis trichome cells. 1099 73

GSH synthesis occurs via two enzymatic steps catalyzed by glutamate-cysteine ligase (GCL, made up of two subunits) and GSH synthetase (GS). Recently, we described coordinate induction of GCL subunits and GS. To study GS transcriptional regulation, we have cloned and characterized a 2.2-kb 5'-flanking region of the rat GS (GenBank accession number AF333982). One transcriptional start site is located at 51 nucleotides upstream of the translational start site. The rat GS promoter drove efficiently luciferase expression in H4IIE cells. Sequential deletion analysis revealed DNA regions that are involved in positive and negative regulation. One repressor identified was NF1. tert-Butylhydroquinone (TBH) exerted a dose- and time-dependent increase in the mRNA level and promoter activity of both GCL subunits and GS. TBH increased protein binding to several regions of the GS promoter, c-jun expression, and activator protein 1 (AP-1) binding activity to several of the putative AP-1-binding sites of the GS promoter. Blocking AP-1 binding with dominant-negative c-jun led to decreased basal expression and significantly blocked the TBH-induced increase in promoter activity and mRNA level of all three genes. In conclusion, AP-1 is required for basal expression of GCL and GS; while NF1 serves as a repressor of GS, increased AP-1 transactivation is the predominant mechanism for coordinate induction of GCL and GS expression by TBH.
J Biol Chem 2002 Sep 20
PMID:Role of AP-1 in the coordinate induction of rat glutamate-cysteine ligase and glutathione synthetase by tert-butylhydroquinone. 1209 5

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.
Plant Physiol 1994 Sep
PMID:Increased Activity of [gamma]-Glutamylcysteine Synthetase in Tomato Cells Selected for Cadmium Tolerance. 1223 24

The thermophilic bacterium Thermus thermophilus synthesizes lysine through the alpha-aminoadipate pathway, which uses alpha-aminoadipate as a biosynthetic intermediate of lysine. LysX is the essential enzyme in this pathway, and is believed to catalyze the acylation of alpha-aminoadipate. We have determined the crystal structures of LysX and its complex with ADP at 2.0A and 2.38A resolutions, respectively. LysX is composed of three alpha+beta domains, each composed of a four to five-stranded beta-sheet core flanked by alpha-helices. The C-terminal and central domains form an ATP-grasp fold, which is responsible for ATP binding. LysX has two flexible loop regions, which are expected to play an important role in substrate binding and protection. In spite of the low level of sequence identity, the overall fold of LysX is surprisingly similar to that of other ATP-grasp fold proteins, such as D-Ala:D-Ala ligase, PurT-encoded glycinamide ribonucleotide transformylase, glutathione synthetase, and synapsin I. In particular, they share a similar spatial arrangement of the amino acid residues around the ATP-binding site. This observation strongly suggests that LysX is an ATP-utilizing enzyme that shares a common evolutionary ancestor with other ATP-grasp fold proteins possessing a carboxylate-amine/thiol ligase activity.
J Mol Biol 2003 Sep 19
PMID:Crystal structure of a lysine biosynthesis enzyme, LysX, from Thermus thermophilus HB8. 1296 79

Skeletal muscle differentation is a complex process regulated at multiple levels. This study addressed the effect of glutathione (GSH) depletion on the transition of murine skeletal muscle C2C12 myoblasts into myocytes induced by growth factor inactivation. Cellular GSH levels increased within 24 hours on myogenic stimulation of myoblasts due to enhanced GSH synthetic rate accounted for by stimulated glutamate-L-cysteine ligase (also known as gamma-glutamylcysteine synthetase) activity. In contrast, the synthesis rate of GSH using gamma-glutamylcysteine and glutamate as precursors, which reflects the activity of the GSH synthetase, did not change during differentiation. The stimulation of GSH stores preceded the myogenic differentiation of C2C12 myoblasts monitored by expression of muscle-specific genes, creatine kinase (CK), myosin heavy chain (MyHC), and MyoD. The pattern of DNA binding activity of NF-kappaB and AP-1 in differentiating cells was similar both displaying an activation peak at 24 hours after myogenic stimulation. Depletion of cellular GSH levels 24 hours after stimulation of differentiation abrogated myogenesis as reflected by lower CK activity, MyHC levels, MyoD expression, and myotubes formation, effects that were reversible on GSH replenishment by GSH ethyl ester (GHSEE). Moreover, GSH depletion led to sustained activation of NF-kappaB, while GSHEE prevented it. Furthermore, inhibition of NF-kappaB activation restored myogenesis despite GSH depletion. Thus, GSH contributes to the formation of myotubes from satellite myoblasts by ensuring inactivation of NF-kappaB, and hence maintaining optimal GSH levels may be beneficial in restoring muscle mass in chronic inflammatory disorders.
Am J Pathol 2004 Sep
PMID:Glutathione depletion impairs myogenic differentiation of murine skeletal muscle C2C12 cells through sustained NF-kappaB activation. 1533 97


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