Phi Class of Glutathione S-transferase Gene Superfamily Widely Exists in Nonplant Taxonomic Groups.
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ABSTRACT:
SUBMITTER: Munyampundu JP
PROVIDER: S-EPMC4750895 | biostudies-literature | 2016
REPOSITORIES: biostudies-literature
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Project description:Glutathione transferases (GSTs) constitute a superfamily of enzymes with essential roles in cellular detoxification and secondary metabolism in plants as in other organisms. Several plant GSTs, including those of the Phi class (GSTFs), require a conserved catalytic serine residue to perform glutathione (GSH)-conjugation reactions. Genomic analyses revealed that terrestrial plants have around ten GSTFs, eight in the Populus trichocarpa genome, but their physiological functions and substrates are mostly unknown. Transcript expression analyses showed a predominant expression of all genes both in reproductive (female flowers, fruits, floral buds) and vegetative organs (leaves, petioles). Here, we show that the recombinant poplar GSTF1 (PttGSTF1) possesses peroxidase activity toward cumene hydroperoxide and GSH-conjugation activity toward model substrates such as 2,4-dinitrochlorobenzene, benzyl and phenetyl isothiocyanate, 4-nitrophenyl butyrate and 4-hydroxy-2-nonenal but interestingly not on previously identified GSTF-class substrates. In accordance with analytical gel filtration data, crystal structure of PttGSTF1 showed a canonical dimeric organization with bound GSH or 2-(N-morpholino)ethanesulfonic acid molecules. The structure of these protein-substrate complexes allowed delineating the residues contributing to both the G and H sites that form the active site cavity. In sum, the presence of GSTF1 transcripts and proteins in most poplar organs especially those rich in secondary metabolites such as flowers and fruits, together with its GSH-conjugation activity and its documented stress-responsive expression suggest that its function is associated with the catalytic transformation of metabolites and/or peroxide removal rather than with ligandin properties as previously reported for other GSTFs.
| S-EPMC4274894 | biostudies-literature
Project description:The evolution and growth of multiple-herbicide resistance (MHR) in grass weeds continues to threaten global cereal production. While various processes can contribute to resistance, earlier work has identified the phi class glutathione-S-transferase (AmGSTF1) as a functional biomarker of MHR in black-grass (Alopecurus myosuroides). This study provides further insights into the role of AmGSTF1 in MHR using a combination of chemical and structural biology. Crystal structures of wild-type AmGSTF1, together with two specifically designed variants that allowed the co-crystal structure determination with glutathione and a glutathione adduct of the AmGSTF1 inhibitor 4-chloro-7-nitro-benzofurazan (NBD-Cl) were obtained. These studies demonstrated that the inhibitory activity of NBD-Cl was associated with the occlusion of the active site and the impediment of substrate binding. A search for other selective inhibitors of AmGSTF1, using ligand-fishing experiments, identified a number of flavonoids as potential ligands. Subsequent experiments using black-grass extracts discovered a specific flavonoid as a natural ligand of the recombinant enzyme. A series of related synthetic flavonoids was prepared and their binding to AmGSTF1 was investigated showing a high affinity for derivatives bearing a O-5-decyl-α-carboxylate. Molecular modelling based on high-resolution crystal structures allowed a binding pose to be defined which explained flavonoid binding specificity. Crucially, high binding affinity was linked to a reversal of the herbicide resistance phenotype in MHR black-grass. Collectively, these results present a nature-inspired new lead for the development of herbicide synergists to counteract MHR in weeds.
| S-EPMC8564858 | biostudies-literature
Project description:In the present work, we report a novel class of glutathione transferases (GSTs) originated from the pathogenic soil bacterium Agrobacterium tumefaciens C58, with structural and catalytic properties not observed previously in prokaryotic and eukaryotic GST isoenzymes. A GST-like sequence from A. tumefaciens C58 (Atu3701) with low similarity to other characterized GST family of enzymes was identified. Phylogenetic analysis showed that it belongs to a distinct GST class not previously described and restricted only in soil bacteria, called the Eta class (H). This enzyme (designated as AtuGSTH1-1) was cloned and expressed in E. coli and its structural and catalytic properties were investigated. Functional analysis showed that AtuGSTH1-1 exhibits significant transferase activity against the common substrates aryl halides, as well as very high peroxidase activity towards organic hydroperoxides. The crystal structure of AtuGSTH1-1 was determined at 1.4 Å resolution in complex with S-(p-nitrobenzyl)-glutathione (Nb-GSH). Although AtuGSTH1-1 adopts the canonical GST fold, sequence and structural characteristics distinct from previously characterized GSTs were identified. The absence of the classic catalytic essential residues (Tyr, Ser, Cys) distinguishes AtuGSTH1-1 from all other cytosolic GSTs of known structure and function. Site-directed mutagenesis showed that instead of the classic catalytic residues, an Arg residue (Arg34), an electron-sharing network, and a bridge of a network of water molecules may form the basis of the catalytic mechanism. Comparative sequence analysis, structural information, and site-directed mutagenesis in combination with kinetic analysis showed that Phe22, Ser25, and Arg187 are additional important residues for the enzyme's catalytic efficiency and specificity.
| S-EPMC3319563 | biostudies-literature
Project description:Fasciola hepatica (liver fluke), a significant threat to food security, causes global economic loss for the livestock industry and is re-emerging as a foodborne disease of humans. In the absence of vaccines, treatment control is by anthelmintics; with only triclabendazole (TCBZ) currently effective against all stages of F. hepatica in livestock and humans. There is widespread resistance to TCBZ and its detoxification by flukes might contribute to the mechanism. However, there is limited phase I capacity in adult parasitic helminths with the phase II detoxification system dominated by the soluble glutathione transferase (GST) superfamily. Previous proteomic studies have demonstrated that the levels of Mu class GST from pooled F. hepatica parasites respond under TCBZ-sulphoxide (TCBZ-SO) challenge during in vitro culture ex-host. We have extended this finding by exploiting a sub-proteomic lead strategy to measure the change in the total soluble GST profile (GST-ome) of individual TCBZ-susceptible F. hepatica on TCBZ-SO-exposure in vitro culture. TCBZ-SO exposure demonstrated differential abundance of FhGST-Mu29 and FhGST-Mu26 following affinity purification using both GSH and S-hexyl GSH affinity. Furthermore, a low or weak affinity matrix interacting Mu class GST (FhGST-Mu5) has been identified and recombinantly expressed and represents a new low-affinity Mu class GST. Low-affinity GST isoforms within the GST-ome was not restricted to FhGST-Mu5 with a second likely low-affinity sigma class GST (FhGST-S2) uncovered. This study represents the most complete Fasciola GST-ome generated to date and has supported the potential of subproteomic analyses on individual adult flukes.
| S-EPMC7889535 | biostudies-literature
Project description:The glutathione transferase (GST) superfamily plays key roles in the detoxification of various xenobiotics. Here, we report the isolation and characterization of a silkworm protein belonging to a previously reported theta-class GST family. The enzyme (bmGSTT) catalyzes the reaction of glutathione with 1-chloro-2,4-dinitrobenzene, 1,2-epoxy-3-(4-nitrophenoxy)-propane, and 4-nitrophenethyl bromide. Mutagenesis of highly conserved residues in the catalytic site revealed that Glu66 and Ser67 are important for enzymatic function. These results provide insights into the catalysis of glutathione conjugation in silkworm by bmGSTT and into the metabolism of exogenous chemical agents.
| S-EPMC4029803 | biostudies-literature
Project description:Mutations in ganglioside-induced differentiation-associated protein 1 (GDAP1) alter mitochondrial morphology and result in several subtypes of the inherited peripheral neuropathy Charcot-Marie-Tooth disease; however, the mechanism by which GDAP1 functions has remained elusive. GDAP1 contains primary sequence homology to the GST superfamily; however, the question of whether GDAP1 is an active GST has not been clearly resolved. Here, we present biochemical evidence, suggesting that GDAP1 has lost the ability to bind glutathione without a loss of substrate binding activity. We have revealed that the α-loop, located within the H-site motif is the primary determinant for substrate binding. Using structural data of GDAP1, we have found that critical residues and configurations in the G-site which canonically interact with glutathione are altered in GDAP1, rendering it incapable of binding glutathione. Last, we have found that the overexpression of GDAP1 in HeLa cells results in a mitochondrial phenotype which is distinct from oxidative stress-induced mitochondrial fragmentation. This phenotype is dependent on the presence of the transmembrane domain, as well as a unique hydrophobic domain that is not found in canonical GSTs. Together, we data point toward a non-enzymatic role for GDAP1, such as a sensor or receptor.
| S-EPMC9394736 | biostudies-literature
Project description:production of glutathione s-transferase from Lactobacillus plantarum Ku720558
| PRJEB33504 | ENA
Project description:Enzyme screens with Strep-tagged recombinant proteins and expression studies with the respective green fluorescent protein (GFP) fusions have been employed to examine the functional activities and subcellular localization of members of the Arabidopsis glutathione transferase (GST) superfamily. Fifty-one of 54 GST family members were transcribed and 41 found to express as functional glutathione-dependent enzymes in Escherichia coli. Functional redundancy was observed and in particular three theta (T) class GSTs showed conserved activities as hydroperoxide-reducing glutathione peroxidases (GPOXs). When expressed in tobacco as GFP fusions, all three GSTTs localized to the peroxisome, where their GPOX activity could prevent membrane damage arising from fatty acid oxidation. Through alternative splicing, two of these GSTTs form fusions with Myb transcription factor-like domains. Examination of one of these variants showed discrete localization within the nucleus, possibly serving a role in reducing nucleic acid hydroperoxides or in signalling. Based on this unexpected differential sub-cellular localization, 15 other GST family members were expressed as GFP fusions in tobacco. Most accumulated in the cytosol, but GSTU12 localized to the nucleus, a family member resembling a bacterial tetrachlorohydroquinone dehalogenase selectively associated with the plasma membrane, and a lambda GSTL2 was partially directed to the peroxisome after removal of a putative chloroplast transit peptide. Based on the results obtained with the GSTTs, it was concluded that these proteins can exert identical protective functions in differing subcellular compartments.
| S-EPMC2657551 | biostudies-literature
Project description:Glutathione S-transferases (GSTs) are dimeric proteins that play a major role in cellular detoxification. The GSTs in mosquito Anopheles dirus species B, an important malaria vector in South East Asia, are of interest because they can play an important role in insecticide resistance. In the present study, we characterized the Anopheles dirus (Ad)GST D3-3 which is an alternatively spliced product of the adgst1AS1 gene. The data from the crystal structure of GST D3-3 shows that Ile-52, Glu-64, Ser-65, Arg-66 and Met-101 interact directly with glutathione. To study the active-site function of these residues, alanine substitution site-directed mutagenesis was performed resulting in five mutants: I52A (Ile-52-->Ala), E64A, S65A, R66A and M101A. Interestingly, the E64A mutant was expressed in Escherichia coli in inclusion bodies, suggesting that this residue is involved with the tertiary structure or folding property of this enzyme. However, the I52A, S65A, R66A and M101A mutants were purified by glutathione affinity chromatography and the enzyme activity characterized. On the basis of steady-state kinetics, difference spectroscopy, unfolding and refolding studies, it was concluded that these residues: (1) contribute to the affinity of the GSH-binding site ('G-site') for GSH, (2) influence GSH thiol ionization, (3) participate in kcat regulation by affecting the rate-limiting step of the reaction, and in the case of Ile-52 and Arg-66, influenced structural integrity and/or folding of the enzyme. The structural perturbations from these mutants are probably transmitted to the hydrophobic-substrate-binding site ('H-site') through changes in active site topology or through effects on GSH orientation. Therefore these active site residues appear to contribute to various steps in the catalytic mechanism, as well as having an influence on the packing of the protein.
| S-EPMC1133834 | biostudies-literature
Project description:Liver fluke infection of livestock causes economic losses of over US$ 3 billion worldwide per annum. The disease is increasing in livestock worldwide and is a re-emerging human disease. There are currently no commercial vaccines, and only one drug with significant efficacy against adult worms and juveniles. A liver fluke vaccine is deemed essential as short-lived chemotherapy, which is prone to resistance, is an unsustainable option in both developed and developing countries. Protein superfamilies have provided a number of leading liver fluke vaccine candidates. A new form of glutathione transferase (GST) family, Sigma class GST, closely related to a leading Schistosome vaccine candidate (Sm28), has previously been revealed by proteomics in the liver fluke but not functionally characterised.In this manuscript we show that a purified recombinant form of the F. hepatica Sigma class GST possesses prostaglandin synthase activity and influences activity of host immune cells. Immunocytochemistry and western blotting have shown the protein is present near the surface of the fluke and expressed in eggs and newly excysted juveniles, and present in the excretory/secretory fraction of adults. We have assessed the potential to use F. hepatica Sigma class GST as a vaccine in a goat-based vaccine trial. No significant reduction of worm burden was found but we show significant reduction in the pathology normally associated with liver fluke infection.We have shown that F. hepatica Sigma class GST has likely multi-functional roles in the host-parasite interaction from general detoxification and bile acid sequestration to PGD synthase activity.
| S-EPMC3362645 | biostudies-literature