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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)

Small metal-binding peptides, cadystins, with the general structure of (gamma-Glu-Cys)n-Gly ((gamma EC)nG), were synthesized in a cell-free system of fission yeast to examine the in vivo synthetic pathway. The crude enzyme for cadystin synthesis was prepared by ammonium sulfate precipitation (75% saturation) from the 120,000 x g supernatant of the cell extract, and the excess salt in the enzyme fraction was removed by Sephadex gel filtration. Using this crude enzyme fraction, it was shown that there were two pathways for cadystin biosynthesis. One pathway is gamma-Glu-Cys (gamma EC) dipeptidyl transfer from both glutathione (gamma ECG) and cadystins to glutathione and cadystins. The other one is gamma EC polymerization from (gamma EC)n and glutathione to (gamma EC)n + i, followed by glycine addition with glutathione synthetase.
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PMID:Two pathways in the biosynthesis of cadystins (gamma EC)nG in the cell-free system of the fission yeast. 203 14

The glutathione synthetase from Escherichia coli B has been crystallized from 27% saturated ammonium sulfate solution (pH 5.5). The crystals are hexagonal, space group P6(2)22 or P6(4)22. The cell dimensions are a = b = 88.0 A, c = 164.2 A, and gamma = 120 degrees. The enzyme is a tetramer (Mr = 143,000) with 222 symmetry, and the asymmetric unit contains one subunit molecule (Mr = 35,600). The crystals diffract to at least 2.5 A resolution.
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PMID:Crystallization and preliminary X-ray studies of glutathione synthetase from Escherichia coli B. 268 23

Glutathione (GSH) synthetase (EC 6.3.2.3) was purified from the fission yeast Schizosaccharomyces pombe L972h- and from the GSH synthetase deficient mutant MN101/pYS41, which harbors a plasmid containing the GSH synthetase gene of the fission yeast. GSH synthetase is expressed at 10 times higher the amount in MN101/pYS41 than in wild-type L972h-. The purified enzyme gave a single band on polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate (native PAGE). The molecular weight of this enzyme was determined to be 1.2 x 10(5) by Sepharose CL-6B gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) revealed that this enzyme was composed of two kinds of subunits, A (M(r) = 33 x 10(3)) and B (M(r) = 26 x 10(3)), and existed as a heterotetramer (A2B2). The enzyme purified from the wild-type fission yeast, which did not harbor the plasmid, showed the same electrophoretic mobilities on both native PAGE and SDS-PAGE and similar catalytic properties under standard conditions. This enzyme is most active at 45 degrees C and pH 8.0-8.5 with 20 mM Mg2+ + 10 mM ATP and 50 mM K+. The strict requirement for the monovalent cation is rather specific for the enzymes from yeasts. The presence of sugar components in the enzyme is also observed, similar to that in the rat kidney enzyme.
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PMID:Glutathione synthetase from the fission yeast. Purification and its unique heteromeric subunit structure. 819 97

The crystal structure of glutathione synthetase from Escherichia coli B complexed with ADP, glutathione, and sulfate has been determined at 2.0 A resolution. Concerning the chemical similarity of sulfate and phosphate, this quaternary complex structure represents a pseudo enzyme-substrate complex in the reverse reaction and consequently allows us to understand the active site architecture of the E. coli glutathione synthetase. Two Mg2+ ions are coordinated with oxygen atoms from the alpha- and beta-phosphate groups of ADP and from the sulfate ion. The flexible loops, invisible in the unliganded or the binary and ternary complex structures, are fixed in the quaternary complex. The larger flexible loop (Ile226-Arg241) includes one turn of a 310-helix that comprises the binding site of the glycine moiety of GSH. The small loop (Gly164-Gly167) is involved in nucleotide binding and acts as a phosphate gripper. The side chains of Arg210 and Arg225 interact with the sulfate ion and the beta-phosphate moiety of ADP. Arg 210 is likely to interact with the carboxylate of the C-terminal gamma-glutamylcysteine in the substrate-binding form of the forward reaction. Other positively charged residues in the active site (Lys125 and Lys160) are involved in nucleotide binding, directing the phosphate groups to the right position for catalysis. Functional aspects of the active site architecture in the substrate-binding form are discussed.
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PMID:A pseudo-michaelis quaternary complex in the reverse reaction of a ligase: structure of Escherichia coli B glutathione synthetase complexed with ADP, glutathione, and sulfate at 2.0 A resolution. 881 Sep 1

The intercellular distribution of the enzymes and metabolites of assimilatory sulfate reduction and glutathione synthesis was analyzed in maize (Zea mays L. cv LG 9) leaves. Mesophyll cells and strands of bundle-sheath cells from second leaves of 11-d-old maize seedlings were obtained by two different mechanical-isolation methods. Cross-contamination of cell preparations was determined using ribulose bisphosphate carboxylase (EC 4.1.1.39) and nitrate reductase (EC 1.6.6.1) as marker enzymes for bundle-sheath and mesophyll cells, respectively. ATP sulfurylase (EC 2.7.7.4) and adenosine 5'-phosphosulfate sulfotransferase activities were detected almost exclusively in the bundle-sheath cells, whereas GSH synthetase (EC 6.3.2.3) and cyst(e)ine, gamma-glutamylcysteine, and glutathione were located predominantly in the mesophyll cells. Feeding experiments using [35S]sulfate with intact leaves indicated that cyst(e)ine was the transport metabolite of reduced sulfur from bundle-sheath to mesophyll cells. This result was corroborated by tracer experiments, which showed that isolated bundle-sheath strands fed with [35S]sulfate secreted radioactive cyst(e)ine as the sole thiol into the resuspending medium. The results presented in this paper show that assimilatory sulfate reduction is restricted to the bundle-sheath cells, whereas the formation of glutathione takes place predominantly in the mesophyll cells, with cyst(e)ine functioning as a transport metabolite between the two cell types.
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PMID:Cyst(e)ine is the transport metabolite of assimilated sulfur from bundle-sheath to mesophyll cells in maize leaves 953 48

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

The complexation and sequestration of heavy metal ions (e.g. Cd) by the cysteine-rich polypeptides known as phytochelatins (PC) are thought to confer heavy metal hyperaccumulation and tolerance in some plant species. PC is synthesized enzymatically from glutathione. The tripeptide glutathione is a product of primary sulfur metabolism. A variety of enzymes or proteins are involved in sulfur assimilation including sulfate transporters (STs), ATP sulfurylase (ATPS), APS reductase (APSR), sulfite reductase (SiR), glutathione synthetase (GS) and phytochelatin synthesis (PCS). These enzymes or proteins are upstream-regulated by Cd at either the metabolic or the genetic level under metal stress. Increasing evidence shows that enhancement of sulfate uptake and reduction occurs with the production of PC in plants under heavy metal stress. In this article, the key aspects of our recent understanding of regulatory mechanisms involved in the relation between the sulfate assimilation and phytochelatin synthesis are described.
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PMID:[Plant sulfate assimilation and regulation of the activity of related enzymes under cadmium stress]. 1647 25

The levels of cysteine (Cys), gamma-glutamylcysteine (gammaEC), and glutathione (GSH) were measured in the endosperms, scutella, roots, and shoots of maize (Zea mays L.) seedlings. GSH was the major thiol in roots, shoots, and scutella, Cys predominated in endosperms. The endosperm, scutellum, and functional phloem translocation were required for maintenance of GSH pools in roots and shoots of 6-day-old seedlings. Exposure of roots to 3 micromolar Cd, besides causing a decline in GSH, caused an accumulation of gammaEC, as if the activity of GSH synthetase was reduced in vivo. [(35)S]Cys injected into endosperms of seedlings was partly metabolized to [(35)S]sulfate. The scutella absorbed both [(35)S]sulfate and [(35)S]Cys and transformed 68 to 87% of the radioactivity into [(35)S]GSH. [(35)S]GSH was translocated to roots and shoots in proportion to the tissue fresh weight. Taken together, the data supported the hypothesis that Cys from the endosperm is absorbed by the scutellum and used to synthesize GSH for transfer through the phloem to the root and shoot. The estimated flux of GSH to the roots was 35 to 60 nanomoles per gram per hour, which totally accounted for the small gain in GSH in roots between days 6 and 7. For Cd-treated roots the GSH influx was similar, yet the GSH pool did not recover to control levels within 24 hours. The estimated flux of GSH to the entire shoot was like that to the roots; however, it was low (11-13 nanomoles per gram per hour) to the first leaf and high (76-135 nanomoles per gram per hour) to the second and younger leaves.
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PMID:Cysteine, gamma-Glutamylcysteine, and Glutathione Levels in Maize Seedlings : Distribution and Translocation in Normal and Cadmium-Exposed Plants. 1666 59

Cysteine, gamma-glutamylcysteine, and glutathione and the extractable activity of the enzymes of glutathione biosynthesis, gamma-glutamylcysteine synthetase (EC 6.3.2.2) and glutathione synthetase (EC 6.3.2.3), were measured in roots and leaves of maize seedlings (Zea mays L. cv LG 9) exposed to CdCl(2) concentrations up to 200 micromolar. At 50 micromolar Cd(2+), gamma-glutamylcysteine contents increased continuously during 4 days up to 21-fold and eightfold of the control in roots and leaves, respectively. Even at 0.5 micromolar Cd(2+), the concentration of gamma-glutamylcysteine in the roots was significantly higher than in the control. At 5 micromolar and higher Cd(2+) concentrations, a significant increase in gamma-glutamylcysteine synthetase activity was measured in the roots, whereas in the leaves this enzyme activity was enhanced only at 200 micromolar Cd(2+). Labeling of isolated roots with [(35)S]sulfate showed that both sulfate assimilation and glutathione synthesis were increased by Cd. The accumulation of gamma-glutamylcysteine in the roots did not affect the root exudation rate of this compound. Our results indicate that maize roots are at least in part autonomous in providing the additional thiols required for phytochelatin synthesis induced by Cd.
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PMID:Effect of Cadmium on gamma-Glutamylcysteine Synthesis in Maize Seedlings. 1666 2

The redox active peptide glutathione is ubiquitous in nature, but some plants also synthesize glutathione analogs in response to environmental stresses. To understand the evolution of chemical diversity in the closely related enzymes homoglutathione synthetase (hGS) and glutathione synthetase (GS), we determined the structures of soybean (Glycine max) hGS in three states: apoenzyme, bound to gamma-glutamylcysteine (gammaEC), and with hGSH, ADP, and a sulfate ion bound in the active site. Domain movements and rearrangement of active site loops change the structure from an open active site form (apoenzyme and gammaEC complex) to a closed active site form (hGSH*ADP*SO(4)(2-) complex). The structure of hGS shows that two amino acid differences in an active site loop provide extra space to accommodate the longer beta-Ala moiety of hGSH in comparison to the glycinyl group of glutathione. Mutation of either Leu-487 or Pro-488 to an Ala improves catalytic efficiency using Gly, but a double mutation (L487A/P488A) is required to convert the substrate preference of hGS from beta-Ala to Gly. These structures, combined with site-directed mutagenesis, reveal the molecular changes that define the substrate preference of hGS, explain the product diversity within evolutionarily related GS-like enzymes, and reinforce the critical role of active site loops in the adaptation and diversification of enzyme function.
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PMID:Structural basis for evolution of product diversity in soybean glutathione biosynthesis. 1994 90


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