Characterization of Ser73 in Arabidopsis thaliana Glutathione S-transferase zeta class

Glutathione S-transferases (GSTs) are ubiquitous detoxifying superfamily enzymes. The zeta class GST from Arabidopsis thaliana (AtGSTZ) can efficiently degrade dichloroacetic acid (DCA), which is a common carcinogenic contaminant in drinking water. Ser73 in AtGSTZ is a conserved residue at Glutathione binding site (G-site). Compared with the equivalent residues in other GSTs, the catalytic and structural properties of Ser73 were poorly investigated. In this article, site-saturation mutagenesis was performed to characterize the detailed role of Ser73. The DCA de.chlorinating (DCA-DC) activity showed that most of the mutants had less than 3% of the wild-type activity, except S73T and $73A showing 43.48% and 21.62% of the wild-type activity, respectively, indicating that position 73 in AtGSTZ showed low mutational substitutability. Kinetic experiments revealed that mutants S73T, $73A, and S73G showed low binding affinity and catalytic efficiency toward DCA, 1.8-, 3.1-, and 10.7- fold increases in KmDcA values and 4.0-, 9.6-, and 34.1- fold decreases in KcatDCA/KmDCA values, respectively, compared to the wild type. Thermostability and refolding experiments showed that the wild type maintalned more thermostability and recovered activity. These results demonstrated the important role of Set73 in catalytic activity and structural stability of the enzyme. Such properties of Set73 could be particularly crucial to the molecular evolution of AtGSTZ and might,therefore, help explain why Ser73 is conserved in all GSTs. This conclusion might provide insights into the directed evolution of the DCA-DC activity of AtGSTZ.     

Functional divergence in the glutathione transferase superfamily in plants. Identification of two classes with putative functions in redox homeostasis in Arabidopsis thaliana

Searches with the human Omega glutathione transferase (GST) identified two outlying groups of the GST superfamily in Arabidopsis thaliana which differed from all other plant GSTs by containing a cysteine in place of a serine at the active site. One group consisted of four genes, three of which encoded active glutathione-dependent dehydroascorbate reductases (DHARs). Two DHARs were predicted to be cytosolic, whereas the other contained a chloroplast targeting peptide. The DHARs were also active as thiol transferases but had no glutathione conjugating activity. Unlike most other GSTs, DHARs were monomeric. The other class of GST comprised two genes termed the Lambda GSTs (GSTLs). The recombinant GSTLs were also monomeric and had glutathione-dependent thiol transferase activity. One GSTL was cytosolic, whereas the other was chloroplast-targeted. When incubated with oxidized glutathione, the putative active site cysteine of the GSTLs and cytosolic DHARs formed mixed disulfides with glutathione, whereas the plastidic DHAR formed an intramolecular disulfide. DHAR S-glutathionylation was consistent with a proposed catalytic mechanism for dehydroascorbate reduction. Roles for the cytosolic DHARs and GSTLs as antioxidant enzymes were also inferred from the induction of the respective genes following exposure to chemicals and oxidative stress.     

Purification and characterization of human muscle glutathione S-transferases: evidence that glutathione S-transferase zeta corresponds to a locus distinct from GST1, GST2, and GST3.

Human muscle glutathione S-transferase isozyme, GST zeta (pI 5.2) has been purified by three different methods using immunoaffinity chromatography, DEAE cellulose chromatography, and isoelectric focusing. GST zeta prepared by any of the three methods does not recognize antibodies raised against the alpha, mu, or pi class glutathione S-transferases of human tissues. GST zeta has a blocked N-terminus and its peptide fingerprints also indicate it to be distinct from the alpha, mu, or pi class isozymes. As compared to GSTs of alpha, mu, and pi classes, GST zeta displays higher activities toward t-stilbene oxide and Leukotriene A4 methyl ester. GST zeta also expresses GSH-peroxidase activity toward hydrogen peroxide. The Kms of GST zeta for CDNB and GSH were comparable to those reported for other human GSTs but its Vmax for CDNB, 7620 mol/mol/min, was found to be considerably higher than that reported for other human GSTs. The kinetics of inhibition of GST zeta by hematin, bile acids, and other inhibitors also indicate that it was distinct from the three classes of GST isozymes. These studies suggest that GST zeta corresponds to a locus distinct from GST1, GST2, and GST3 and probably corresponds to the GST4 locus as suggested previously by Laisney et al. (1984, Human Genet. 68, 221-227). The results of peptide fingerprints and kinetic analysis indicate that as compared to the pi and alpha class isozymes, GST zeta has more structural and functional similarities with the mu class isozymes. Besides GST zeta several other GST isozymes belonging to pi and mu class have also been characterized in muscle. The pi class GST isozymes of muscle have considerable charge heterogeneity among them despite identical N-terminal sequences.     

Proteomic Analysis of Glutathione S-Transferases of Arabidopsis thaliana Reveals Differential Salicylic Acid-Induced Expression of the Plant-Specific Phi and Tau Classes

Plant glutathione S -transferases (GSTs) are a large group of multifunctional proteins that are induced by diverse stimuli. Using proteomic approaches we identified 20 GSTs at the protein level in Arabidopsis cell culture with a combination of GST antibody detection, LC-MS/MS analysis of 23-30 kDa proteins and glutathione-affinity chromatography. GSTs identified were from phi, tau, theta, zeta and DHAR sub-sections of the GST superfamily of 53 members. We have uncovered preliminary evidence for post-translational modifications of plant GSTs and show that phosphorylation is unlikely to be responsible. Detailed analysis of GST expression in response to treatment with 0.01-1 mM of the plant defence signal salicylic acid (SA) uncovered some interesting features. Firstly, GSTs appear to display class-specific concentration-dependent SA induction profiles highlighting differences between the large, plant specific phi and tau classes. Secondly, different members of the same class, while sharing similar SA dose responses, may display differences in terms of magnitude and timing of induction, further highlighting the breadth of GST gene regulation. Thirdly, closely related members of the same class ( GSTF6 and GSTF7 ), arising via tandem duplication, may be regulated differently in terms of basal expression levels and also magnitude of induction raising questions about the role of subfunctionalisation within this family. Our results reveal that GSTs exhibit class specific responses to SA treatment suggesting that several mechanisms are acting to induce GSTs upon SA treatment and hinting at class-specific functions for this large and important, yet still relatively elusive gene family.     

Induction of glutathione S-transferases in Arabidopsis by herbicide safeners

Herbicide safeners increase herbicide tolerance in cereals but not in dicotyledenous crops. The reason(s) for this difference in safening is unknown. However, safener-induced protection in cereals is associated with increased expression of herbicide detoxifying enzymes, including glutathione S-transferases (GSTs). Treatment of Arabidopsis seedlings growing in liquid medium with various safeners similarly resulted in enhanced GST activities toward a range of xenobiotics with benoxacor, fenclorim, and fluxofenim being the most effective. Safeners also increased the tripeptide glutathione content of Arabidopsis seedlings. However, treatment of Arabidopsis plants with safeners had no effect on the tolerance of seedlings to chloroacetanilide herbicides. Each safener produced a distinct profile of enhanced GST activity toward different substrates suggesting a differential induction of distinct isoenzymes. This was confirmed by analysis of affinity-purified GST subunits by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. AtGSTU19, a tau class GST, was identified as a dominant polypeptide in all samples. When AtGSTU19 was expressed in Escherichia coli, the recombinant enzyme was highly active toward 1-chloro-2,4-dinitrobenzene, as well as chloroacetanilide herbicides. Immunoblot analysis confirmed that AtGSTU19 was induced in response to several safeners. Differential induction of tau GSTs, as well as members of the phi and theta classes by safeners, was demonstrated by RNA-blot analysis. These results indicate that, although Arabidopsis may not be protected from herbicide injury by safeners, at least one component of their detoxification systems is responsive to these compounds.     

Inhibitory effects of auxins and related substances on the activity of an Arabidopsis glutathione S-transferase isozyme expressed in Escherichia coli

Glutathione S-transferases (GSTs; EC 2.5.1.18) are encoded by a gene family. Some GSTs have the capacity to bind to indole-3-acetic acid (IAA), whereas the gene expression of other GSTs is regulated by auxin. In order to assess a possible physiological significance of the auxin binding of GST, we investigated effects of auxins on the activity of GST expressed in Escherichia coli. cDNA cloning was carried out for the fifth gene ( GST5 ) of GST in Arabidopsis. Although the deduced amino acid sequence of GST5 was remotely related to that of the other Arabidopsis GSTs (less than 20% identical), the GST5 protein (GST5) expressed in E. coli showed GST activity. Apparent K_m values of GST5 are 0.86 and 1.29 m M for glutathione (GSH) and 1-chloro-2,4-dinitrobenzene, respectively. IAA, 2,4-dichlorophenoxyacetic acid (2,4-D), 1-naphthaleneacetic acid (1-NAA) and 2-NAA inhibited the enzyme activity competitively with respect to GSH. The apparent K_i of IAA is 1.56 m M . Salicylic acid inhibited GST activity in a noncompetitive manner. 2,4-D was the most inhibitory among the tested chemicals. GST5 bound to GSH-immobilized agarose gel was effectively eluted by IAA. These results indicate that IAA and the related substances bind to GST5 at the GSH-binding site, and exclude the possibility that the compounds could be substrates for GST5. Although the K_i value of IAA is too high for any physiological consequences, it might be assumed that GST activity is modulated in vivo by an auxin-related substance(s). The steady-state level of the GST5 mRNA was increased by wounding, heat shock, and spraying buffer on the plant, but was not influenced by auxin treatment.     

Functional characterisation of ganglioside-induced differentiation-associated protein 1 as a glutathione transferase

Mutations in the ganglioside-induced differentiation-associated protein 1 (GDAP1) gene have been linked with Charcot-Marie-Tooth (CMT) disease. This protein, and its paralogue GDAP1L1, appear to be structurally related to the cytosolic glutathione S-transferases (GST) including an N-terminal thioredoxin fold domain with conserved active site residues. The specific function, of GDAP1 remains unknown. To further characterise their structure and function we purified recombinant human GDAP1 and GDAP1L1 proteins using bacterial expression and immobilised metal affinity chromatography. Like other cytosolic GSTs, GDAP1 protein has a dimeric structure. Although the full-length proteins were largely insoluble, the deletion of a proposed C-terminal transmembrane domain allowed the preparation of soluble protein. The purified proteins were assayed for glutathione-dependent activity against a library of 'prototypic' GST substrates. No evidence of glutathione-dependent activity or an ability to bind glutathione immobilised on agarose was found.     

2,4-Dichlorophenoxyacetic Acid and Related Chlorinated Compounds Inhibit Two Auxin-Regulated Type-III Tobacco Glutathione S-Transferases

Two auxin-inducible glutathione S-transferase (GST, EC 2.5.1.18) isozymes from tobacco (Nicotiana tabacum, White Burley) were partially characterized. GST1-1 and GST2-1 are members of a recently identified new type of plant GST isozymes that we will here refer to as type III. Both enzymes were active, with 1-chloro-2,4-dinitrobenzene as a substrate, when expressed in bacteria as fusion proteins. The apparent Km for 1-chloro-2,4-dinitrobenzene was found to be 0.85 [plus or minus] 0.25 mM for GST1-1 and 0.20 [plus or minus] 0.15 mM for GST2-1. The apparent Km for glutathione was similar for both enzymes, 0.40 [plus or minus] 0.15 mM. The in vitro activity of both enzymes could be inhibited by the synthetic auxin 2,4-dichlorophenoxyacetic acid, with an apparent Ki of 80 [plus or minus] 40 [mu]M for GST1-1 and 200 [plus or minus] 100 [mu]M for GST2-1. The GST1-1 was also inhibited by structurally related substances, such as 2,4-dichlorobenzoic acid, with a roughly similar Ki. The nonchlorinated structures benzoic acid and phenoxyacetic acid did not inhibit. p-Chloroisobutyric acid, or clofibric acid, an auxin-transport inhibitor, was found to be an active inhibitor as well. The strongest inhibitor identified, however, was a phenylacetic acid derivative, ethacrynic acid, which showed an apparent Ki of 5 [plus or minus] 5 [mu]M for both enzymes. This substance is a known inducer as well as a substrate of specific mammalian GSTs. The results presented here indicate that the type III plant GSTs might be involved in the metabolism or transport of chlorinated substances that are structurally related to auxins. The possibility that auxins are endogenous ligands or substrates for GSTs is discussed.     

Purification and characterization of a cytosolic transglutaminase from a cultured human tumour-cell line.

The cytosolic glutathione transferases (GSTs) with basic pI values have been studied in mouse liver after treatment with 2,3-t-butylhydroxyanisole (BHA), cafestol palmitate (CAF), phenobarbital (PB), 3-methylcholanthrene (3-MC) and trans-stilbene oxide (t-SBO). The cytosolic GST activity was induced by all compounds except for 3-MC. Three forms of GST were isolated by means of affinity chromatography and f.p.l.c. The examination of protein profiles and enzymic activities with specific substrates showed that the three GSTs correspond to those found in control animals, i.e. GSTs MI, MII and MIII. The class Mu GST MIII accounted for the major effect of induction, whereas the class Alpha GST MI and the class Pi GST MII were unchanged or somewhat down-regulated. The greatest induction was obtained with BHA, PB and CAF. The activities of other glutathione-dependent enzymes were also studied. An increase in glutathione reductase and thioltransferase activities was observed after BHA, PB or CAF treatment; glyoxalase I and Se-dependent glutathione peroxidase were depressed in comparison with the control group in all cases studied.     

Purification of a phi-type glutathione S-transferase from pumpkin flowers,and molecular cloning of its cDNA

A major species of glutathione S-transferase (GST), Pugf, was highly purified from pumpkin flowers. Two-dimensional electrophoresis of the purified enzyme gave two adjacent protein spots. The specific activity of the purified enzyme was 2.4 micromol min(-1) mg(-1) protein for 1-chloro-2,4-dinitrobenzene. This value is one to two orders of magnitude lower than that of pumpkin tau-type GSTs. The expression pattern of Pugf in healthy pumpkin plants and responses to various stresses were examined by western blotting. Pugf was found in high concentrations in petioles, stems, and roots as well as flowers, and was more abundant in expanding young organs than in fully expanded mature organs. Dehydration caused a slight increase in its concentration, but high and low temperatures, salty stress, and 2,4-dichlorophenoxyacetic acid seemed to have no effects. A cDNA encoding Pugf was cloned and sequenced. Sequence comparison with other plant GSTs suggested that it should be classified as a phi-type GST.     

Characterization of a novel alpha-class anionic glutathione S-transferase isozyme from human liver

A novel, alpha-class glutathione S-transferase (GST) isozyme has been isolated from human liver using glutathione (GSH) affinity chromatography, DEAE-cellulose ion-exchange chromatography, and immunoaffinity chromatography. The isozyme is a dimer of approximately 25,000 Mr with blocked N termini. Structural, kinetic, and immunological properties of this enzyme indicate that it belongs to the alpha class of GSTs. Noticeable differences between the properties of this enzyme and the other alpha-class GSTs of human liver are its anionic nature (pI 5.0), GSH peroxidase activity toward hydrogen peroxide, and relatively higher GSH conjugating activities toward CDNB and epoxide substrates as compared to other alpha-class GSTs. Results of these studies indicate that anionic GST omega characterized previously (Y. C. Awasthi, D. D. Dao, and R. P. Saneto, 1980, Biochem. J. 191, 1-10) from human liver is a mixture of GST pi and a novel alpha-class GST. We have, therefore, reassigned the name GST omega to this new alpha-class anionic GST of human liver.     

Paragonimus westermani: a cytosolic glutathione S-transferase of a sigma-class in adult stage

We purified cytosolic glutathione S-transferase (GST) of adult Paragonimus westermani monitoring its activity with 1-chloro-2,4-dinitrobenzene (CDNB). The enzyme was purified 18.4-fold to electrophoretic homogeneity with 21% recovery rate through a three-step procedure. The purified enzyme (Pw28GST) has a subunit molecular weight of 28 kDa with an isoelectric point at 4.6. Monoclonal antibody (anti-Pw28GST) against Pw28GST did not cross-react with GSTs from other helminths. cDNA library was constructed in lambdaZAP II bacteriophage and screened with anti-Pw28GST. The corresponding gene containing a single open reading frame of 804 bp encoded 211 amino acids. The predicted amino acid sequence exhibited a higher homology with catalytic domain near N-terminus of class sigma GSTs (58%) than with schistosome 28-kDa GSTs (45-41%) or with class sigma GSTs themselves (33-31%). The sequence contained both Tyr-6 and Tyr-10 that are highly conserved in mammalian and helminth GSTs. The apparent K(m) value of a recombinant enzyme was 0.78 mM. Both native and recombinant enzymes showed the highest activity against CDNB, relatively weak activity against ethacrynic acid and reactive carbonyls, and no activity against epoxy-3-(p-nitrophenoxy)-propane. The activities were inhibited by bromosulfophthalein, cibacron blue, and albendazole, but not by praziquantel. These findings indicate that adult P. westermani has a class sigma GST.     

Purification and characterization of a glutathione S-transferase from the fungus Cunninghamella elegans

Cunninghamella elegans grown on Sabouraud dextrose broth had glutathione S-transferase (GST) activity. The enzyme was purified 172-fold from the cytosolic fraction (120000 x g) of the extract from a culture of C. elegans, using Q-Sepharose ion exchange chromatography and glutathione affinity chromatography. The GST showed activity against 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, 4-nitrobenzyl chloride, and ethacrynic acid. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel filtration chromatography revealed that the native enzyme was homodimeric with a subunit of M(r) 27000. Comparison by Western blot analysis implied that this fungal GST had no relationship with mammalian alpha-, mu-, and pi-class GSTs, although it showed a small degree of cross-reactivity with a theta-class GST. The N-terminal amino acid sequence of the purified enzyme showed no significant homology with other known GSTs.     

The tyrosine kinase activity of the epidermal-growth-factor receptor is necessary for phospholipase A2 activation.

Cytosolic glutathione transferases (GSTs) were purified from the rat spleen by S-hexyl-GSH-Sepharose chromatography, and two major forms were identified as GSTs 2-2 and 7-7 (GST P). Besides these forms an acidic form (pI 5.8) was purified by chromatofocusing at pH 7-4 and it accounted for about 1% of the total GST activity bound to S-hexyl-GSH-Sepharose. Two-dimensional gel electrophoresis revealed that it is a homodimer (subunit Mr 26,000 with pI 5.8). Immunoblot analysis demonstrated that it was immunologically related to GSTs 2-2 and 1-1, and its N-terminal amino acid was apparently blocked, similarly to other forms of the class Alpha. This form had a low activity towards cumene hydroperoxide or 4-hydroxynon-2-enal, indicating that this form differed from GSTs 10-10 and 8-8 as well as from GSTs 1-1 and 2-2. These results suggest that it is a new form of GST belonging to the class Alpha.     

A Nicotiana plumbaginifolia protein labeled with an azido cytokinin agonist is a glutathione S‐transferase

The mechanisms of reception/transduction of cytokinins still remain largely unknown. We used 1‐(2‐azido‐6‐chloropyrid‐4‐yl)‐3‐(4‐[³H])phenylurea ([³H]azido‐CPPU), a new photoaffinity probe to search for cytokinin‐binding proteins. A soluble protein that binds phenylurea‐type cytokinins has been specifically photolabeled in Nicotiana plumbaginifolia (cv. Viviani line pbH1D) leaf extracts. The protein was purified to homogeneity by affinity chromatography. Its N‐terminal amino acid sequence, as well as four internal peptidic sequences are highly homologous with the theta class of the glutathione S‐transferase superfamily (GST, EC 2.5.1.18) including Hyoscyamus muticus and Arabidopsis GSTs identified as auxin‐binding proteins. The purified N. plumbaginifolia protein also possesses GST enzymatic activity. To test the possible involvement of this GST in the mechanism of action of cytokinin, we studied the binding of tritiated‐CPPU to the purified GST in the presence of various compounds, cytokinin agonists, cytokinin antagonists, or inactive molecules. Thidiazuron is a poor competitor, and neither zeatin nor the active optical isomer R‐MeBA is able to inhibit the binding of CPPU. There is no correlation between the cytokinin activity and the binding properties of the molecules tested. Our results confirmed that plant GSTs bind different compounds, especially plant hormones but probably have no specific role in the mode of action of cytokinins.     

Glutathione S-transferases in the Japanese quail: Tissue distribution and purification of the liver isozymes

Cytosolic glutathione S-transferase (GST) activities toward 1-chloro-2,4-dinitro-benzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), ethacrynic acid (EA), 1,2-epoxy-3-(p-nitrophenoxyl)propane (EPNP), trans-4-phenyl-3-buten-2-one (t-PBO), δ³-androstene-3,17-dione (ASD) and trans-stilbene oxide (t-SO); cytosolic glutathione peroxidase activity toward cumene hydroperoxide (CuOOH); and microsomal GST activity toward CDNB were examined in liver, kidney, brain, and lung of adult male and female Japanese quail. In all cases, the renal specific activity per milligram protein was higher than the hepatic activity and was the highest among the four tissues examined. No consistent sex differences in GST activity were observed. The GSTs were purified from quail liver cytosol by S-hexylglutathione and glutathione affinity chromatography. Total GSTs eluted from the S-hexylglutathione affinity column were further separated by chromatofocusing, and the microheterogeneity of the GST isozymes was shown by high-resolution native isoelectrofocusing (IEF) in polyacrylamide slab gels and by SDS-PAGE. Five subunits were identified: QL1 (30.5 kDa), QL2 (27.2 kDa), QL3a (26.8 kDa), QL3b (26.5 kDa), and QL4 (25.5 kDa). Western blot analysis revealed that QL1 and QL2 reacted with antibodies raised against the rat Mu class GSTs (Yb1 and Yb2), and QL3a and QL3b reacted with those raised against the Alpha class (rat Ya and mouse a). Substrate specific activity of each isoform was determined with CDNB, DCNB, CuOOH, EA, t-PBO, ASD, and t-SO. QL3a and QL3b have high reactivity toward CuOOH, while QL1 and QL2 showed high activity toward t-SO. The N-terminal amino acid sequence of QL2 was identical to that of the chicken Mu class GST subunit CL2. However, no sequence was obtained with QL1 due to possible N-terminal blockage. © 1996 John Wiley & Sons, Inc.