Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Identification of a putative flexible loop in Arabidopsis glutathione synthetase.

ABSTRACT: Glutathione synthetase catalyses the ATP-dependent ligation of gamma-glutamylcystene with glycine to form glutathione. Amino acid sequence comparisons between the Arabidopsis and the Escherichia coli proteins suggested that a region, identified as a small flexible loop that covers the active site of the E. coli protein, might be conserved in the eukaryotic protein. Three site-directed mutations in the Arabidopsis protein were generated to test this hypothesis. Two mutations within the conserved region (Lys367/ Pro368-->Asn/Ser and Gly374-->Val) inactivated the enzyme in an in vivo assay based on cadmium resistance in S. pombe, and in an in vitro assay of the activity of the enzyme expressed in E. coli. A third mutation outside of this conserved region (Leu363-->Glu) had a smaller effect in both assays. These results are consistent with the idea that this glycine-rich loop in the Arabidopsis and E. coli proteins might serve the same function in covering the active site of the enzyme.

SUBMITTER: Wang CL

PROVIDER: S-EPMC1218183 | biostudies-other | 1997 Feb

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

Glutathione synthetase: similarities of the proteins from Schizosaccharomyces pombe and Arabidopsis thaliana.

Project description: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.

| S-EPMC1218705 | biostudies-other

Rice plants have three homologs of glutathione synthetase genes, one of which, OsGS2, codes for hydroxymethyl-glutathione synthetase.

Project description:Glutathione is a ubiquitous thiol tripeptide in land plants, and glutathione-like tripeptides can also be found in some plant species. Rice (Oryza sativa) plants synthesize hydroxymethyl-glutathione, in which the terminal glycine residue of glutathione is replaced by a serine residue; however, the biosynthetic pathway of hydroxymethyl-glutathione has not been identified. We isolated three rice glutathione synthetase homologs, designated OsGS1, OsGS2, and OsGS3, and found that knockdown of OsGS2 via RNA interference markedly decreased hydroxymethyl-glutathione concentration in rice plants. The in vitro enzyme assay, using purified recombinant protein, demonstrated that OsGS2 catalyzed the synthesis of hydroxymethyl-glutathione from ?-glutamylcysteine (?EC) and L-serine in an ATP-dependent manner. OsGS2 could also utilize glycine as a cosubstrate with ?EC, but the enzyme-substrate affinity for L-serine was tenfold higher than that for glycine. These results indicate that OsGS2 codes for hydroxymethyl-glutathione synthetase.

| S-EPMC6508825 | biostudies-literature

Genetic Mutations in the S-loop of Human Glutathione Synthetase: Links Between Substrate Binding, Active Site Structure and Allostery.

Project description:The second step in the biosynthesis of the cellular antioxidant glutathione (GSH) is catalyzed by human glutathione synthetase (hGS), a negatively cooperative homodimer. Patients with mutations in hGS have been reported to exhibit a range of symptoms from hemolytic anemia and metabolic acidosis to neurological disorders and premature death. Several patient mutations occur in the S-loop of hGS, a series of residues near the negatively cooperative ?-GC substrate binding site. Experimental point mutations and molecular dynamic simulations show the S-loop not only binds ?-GC through a salt bridge and multiple hydrogen bonds, but the residues also modulate allosteric communication in hGS. By elucidating the role of S-loop residues in active site structure, substrate binding, and allostery, the atomic level sequence of events that leads to the detrimental effects of hGS mutations in patients are more fully understood.

| S-EPMC6297838 | biostudies-literature

Structure of Trypanosoma brucei glutathione synthetase: domain and loop alterations in the catalytic cycle of a highly conserved enzyme.

Project description:Glutathione synthetase catalyses the synthesis of the low molecular mass thiol glutathione from l-gamma-glutamyl-l-cysteine and glycine. We report the crystal structure of the dimeric enzyme from Trypanosoma brucei in complex with the product glutathione. The enzyme belongs to the ATP-grasp family, a group of enzymes known to undergo conformational changes upon ligand binding. The T. brucei enzyme crystal structure presents two dimers in the asymmetric unit. The structure reveals variability in the order and position of a small domain, which forms a lid for the active site and serves to capture conformations likely to exist during the catalytic cycle. Comparisons with orthologous enzymes, in particular from Homo sapiens and Saccharomyces cerevisae, indicate a high degree of sequence and structure conservation in part of the active site. Structural differences that are observed between the orthologous enzymes are assigned to different ligand binding states since key residues are conserved. This suggests that the molecular determinants of ligand recognition and reactivity are highly conserved across species. We conclude that it would be difficult to target the parasite enzyme in preference to the host enzyme and therefore glutathione synthetase may not be a suitable target for antiparasitic drug discovery.

| S-EPMC2845819 | biostudies-literature

Identification of putative Arabidopsis DEMETER target genes by GeneChip analysis.

Project description:The Arabidopsis DEMETER (DME) DNA glycosylase is required for the maternal allele expression of imprinted Polycomb group (MEDEA and FIS2) and transcription factor (FWA) genes in the endosperm. Expression of DME in the central cell, not in pollen or stamen, establishes gene imprinting by hypomethylating maternal alleles. However, little is known about other genes regulated by DME. To identify putative DME target genes, we generated CaMV:DME plants which ectopically express DME in pollen and stamens. Comparison of mRNA profiles revealed 94 genes induced by ectopic DME expression in both stamen and pollen. Gene ontology analysis identified three molecular functions enriched in the DME-inducible RNA list: DNA or RNA binding, kinase activity, and transcription factor activity. Semi-quantitative RT-PCR verified the candidate genes identified by GeneChip analysis. The putative target genes identified in this study will provide insights into the regulatory mechanism of DME DNA glycosylase and the functions of DNA demethylation.

| S-EPMC2137929 | biostudies-literature

Site directed mutagenesis of Schizosaccharomyces pombe glutathione synthetase produces an enzyme with homoglutathione synthetase activity.

Project description:Three different His-tagged, mutant forms of the fission yeast glutathione synthetase (GSH2) were derived by site-directed mutagenesis. The mutant and wild-type enzymes were expressed in E. coli DH5α and affinity purified in a two-step procedure. Analysis of enzyme activity showed that it was possible to shift the substrate specificity of GSH2 from Gly (k(m) 0,19; wild-type) to β-Ala or Ser. One mutation (substitution of Ile471, Cy472 to Met and Val and Ala 485 and Thr486 to Leu and Pro) increased the affinity of GSH2 for β-Ala (k(m) 0,07) and lowered the affinity for Gly (k(m) 0,83), which is a characteristic of the enzyme homoglutathione synthetase found in plants. Substitution of Ala485 and Thr486 to Leu and Pro only, increased instead the affinity of GSH2 for Ser (k(m) 0,23) as a substrate, while affinity to Gly was preserved (k(m) 0,12). This provides a new biosynthetic pathway for hydroxymethyl glutathione, which is known to be synthesized from glutathione and Ser in a reaction catalysed by carboxypeptidase Y. The reported findings provide further insight into how specific amino acids positioned in the GSH2 active site facilitate the recognition of different amino acid substrates, furthermore they support the evolutionary theory that homoglutathione synthetase evolved from glutathione synthetase by a single gene duplication event.

| S-EPMC3473041 | biostudies-literature

Identification of putative Arabidopsis DEMETER target genes by GeneChip Analysis

Project description:We used Affymetrix Arabidopsis ATH1 GeneChip to profile RNAs active in wild type columbia (glabrous) and CaMV::DME pollen and stamens. Keywords: genetic modification

2007-11-01 | GSE9408 | GEO

Glutathione biosynthesis in Arabidopsis trichome cells.

Project description: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.

| S-EPMC27156 | biostudies-literature

Human hereditary glutathione synthetase deficiency: kinetic properties of mutant enzymes.

Project description:Patients with hereditary glutathione synthetase deficiency suffer from haemolytic anaemia, 5-oxoprolinuria, metabolic acidosis, recurrent bacterial infections and various degrees of central nervous system dysfunction. To investigate the molecular basis of the mutations associated with this disease, seven naturally occurring missense mutations [L188P (Leu188-->Pro), D219A, D219G, Y270C, Y270H, R283C and P314L] were expressed using a His-tagged, Escherichia coli-based expression system. Effects of the mutations on kinetic properties, including negative co-operative binding of gamma-glutamyl substrate, were evaluated. The mutation P314L did not have any major effect on these parameters and was classified as a neutral mutation. The remaining mutations decreased V(max) to 2-27% of wild-type activity. Negative co-operativity for gamma-gluABA (L-gamma-glutamyl-L-alpha-aminobutyric acid) was abolished in five mutant recombinant enzymes, whereas for one mutant enzyme, this co-operativity changed from negative to positive. The structural consequences of the mutations were interpreted on the basis of the known structure of the wild-type enzyme.

| S-EPMC1133856 | biostudies-literature

The role of the glycine triad in human glutathione synthetase.

Project description:Experimental kinetics and computational modeling of human glutathione synthetase (hGS) support the significant role of the G-loop glycine triad (G369, G370, G371) for activity of this ATP-grasp enzyme. Enzyme kinetic experiments indicate that G369V and G370V mutant hGS have little activity (<0.7 and 0.3%, respectively, versus wild-type hGS). However, G371V retains ?13% of the activity of wild-type hGS. With respect to G-loop:A-loop interaction in hGS, mutations at Gly369 and Gly370 decrease ligand binding and prevent active site closure and protection. This research indicates that Gly369 and Gly370 have essential roles in hGS, while Gly371 has a lesser involvement. Implications for glycine-rich ensembles in other phosphate-binding enzymes are discussed.

| S-EPMC3065366 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data