Roles for glutathione transferases in plant secondary metabolism

Plant glutathione transferases (GSTs) are classified as enzymes of secondary metabolism, but while their roles in catalysing the conjugation and detoxification of herbicides are well known, their endogenous functions are largely obscure. Thus, while the presence of GST-derived S-glutathionylated xenobiotics have been described in many plants, there is little direct evidence for the accumulation of similarly conjugated natural products, despite the presence of a complex and dichotomous metabolic pathway which processes these reaction products. The conservation in glutathione conjugating and processing pathways, the co-regulation of GSTs with inducible plant secondary metabolism and biochemical studies showing the potential of these enzymes to conjugate reactive natural products are all suggestive of important endogenous functions. As a framework for addressing these enigmatic functions we postulate that either: (a) the natural reaction products of GSTs are unstable and undergo reversible S-glutathionylation; (b) the conjugation products of GSTs are very rapidly processed to derived metabolites; (c) GSTs do not catalyse conventional conjugation reactions but instead use glutathione as a cofactor rather than co-substrate; or (d) GSTs are non-catalytic and function as transporter proteins for secondary metabolites and their unstable intermediates. In this review, we describe how enzyme biochemistry and informatics are providing clues as to GST function allowing for the critical evaluation of each of these hypotheses. We also present evidence for the involvement of GSTs in the synthesis of sulfur-containing secondary metabolites such as volatiles and glucosinolates, and the conjugation, transport and storage of reactive oxylipins, phenolics and flavonoids.     

A ligand function of glutathione S-transferase

Glutathione S-transferases (GSTs) are ubiquitous enzymes and abundant in plants. They are intimately involved in plant metabolism and stress defense related to reactive oxygen species. Our project assigned particular reactions including novel ones to certain GST-isoforms. Transformed E. coli was used to express recombinant GST-isoforms from maize. An N-terminal His tag allowed their purification by affinity chromatography. Three GST-monomers had a molecular weight of 26, 27, 29 kDa, and aggregated to dimers when assayed for their enzymic properties. Four dimeric isoforms were used to study how they interact with tetrapyrroles (of the chlorophyll biosynthesis pathway). It was found that protoporphyrin IX (Proto IX), Mg-protoporphyrin and other tetrapyrroles are bound non-covalently ("liganded") to GSTs but not conjugated with reduced glutathione. This binding is non-covalent, and results in inhibition of conjugation activity, the degree depends on type of the porphyrin and GST-isoform. I50-values between 1-10 microM were measured for Proto IX, the inhibition by mesoporphyrin and Mg-protoporphyrin was 2- to 5-fold less. The ligand binding is noncompetitive for the substrate 1-chloro-2,4-dinitrobenzene and competitive for glutathione. The dimer GST 26/26 prevents the (non-enzymic) autoxidation of protoporphyrinogen to Proto IX, which produces phytotoxic reactive oxygen species in the light. GST 27/27 protects hemin against degradation. Protoporphyrinogen is formed in the plastid and then exported into the cytosol. Apparently binding by a suitable GST-isoform ensures that the highly autoxidizable protoporphyrinogen can safely reach the mitochondrium where it is processed to cytochrome.     

Binding and protection of porphyrins by glutathione S-transferases of Zea mays L

Glutathione S-transferases (GSTs) are multi-functional enzymes, known to conjugate xenobiotics and degrade peroxides. Herein, we report on the potential of four Zea mays GST isoforms (Zm GST I-I, Zm GST I-II, Zm GST II-II and Zm GST III-III) to act as binding and protection proteins. These isoforms bind protoporphyrin IX (PPIX), mesoporphyrin, coproporphyrin, uroporphyrin and Mg-protoporpyhrin, but do not form a glutathione conjugate. The binding is non-covalent and inhibits GSTs enzymatic activity, dependent on the type of the porphyrin and GST isoform tested. I(50) values are in the range of 1 to 10 microM for PPIX, the inhibition by mesoporphyrin and Mg-protoporphyrin (Mg-PPIX) is two to five times less. The mode of binding is non-competitive for the hydrophobic substrate and competitive for glutathione. Binding affinities (K(D) values) of the GST isoforms are between 0.3 and 0.8 microM for coproporphyrin and about 2 microM for mesoporphyrin.Zm GST III-III prevents the nonenzymatic autoxidation of protoporphyrinogen to the phytotoxic PPIX. Zm GST II-II can reduce the oxidative degradation of hemin. This points to a specific ligand role of distinct GST isoforms to protect tetrapyrroles in the plant cell.     

Three distinct-type glutathione S-transferases from Escherichia coli important for defense against oxidative stress

Previously, we characterized glutathione S-transferase (GST) B1-1 from Escherichia coli enzymologically and structurally. Besides GST B1-1, E. coli has seven genes that encode GST-like proteins, for which, except SspA, neither biological roles nor biochemical properties are known. Here we show that the GST-like YfcF and YfcG proteins have low but significant GSH-conjugating activity toward 1-chloro-2,4-dinitorobenzene and GSH-dependent peroxidase activity toward cumene hydroperoxide. Analysis involving site-directed mutagenesis suggested that Ser16 and Asn11 were important for the activities of YfcF and YfcG, respectively. On the contrary, no residue around the catalytic site of GST B1-1 has been demonstrated to be essential for catalytic activity. Deletions of the gst, yfcF, and yfcG genes each decreased the resistibility of the bacteria to hydrogen peroxide, which was recovered by transformation with the expression plasmid for the deleted enzyme. The inactive YfcF(S16G) and YfcG(N11A) mutants, however, could not rescue the knockout bacteria. Thus, E. coli has at least three GSTs of distinct classes, all of which are important for defense against oxidative stress in spite of the structural diversity. This seems consistent with the hypothesis that GSTs constitute a protein superfamily that has evolved from a thioredoxin-like ancestor in response to the development of oxidative stress.     

Purification,regulation and cloning of a glutathione transferase (GST) from maize resembling the auxin-inducible type-III GSTs

The glutathione transferases (GSTs) from maize (Zea mays L.) with activities toward the chloroacetanilide herbicide metolachlor and the diphenyl ether herbicide fluorodifen were fractionated into two pools based on binding to affinity columns. Pool 1 GSTs were retained on Orange A agarose and were identified as isoenzymes Zea mays (Zm) GST I-I, Zm GST I-II and Zm GST I-III, which have been described previously. Pool 2 GSTs selectively bound to S-hexyl-glutathione-Sepharose and were distinct from the pool 1 GSTs, being composed of a homodimer of 28.5 kDa subunits, termed Zm GST V-V, and a heterodimer of the 28.5 kDa polypeptide and a 27.5 kDa subunit, termed Zm GST V-VI. Using an antibody raised to Zm GST V-VI, a cDNA expression library was screened and a Zm GST V clone identified showing sequence similarity to the type-III auxin-inducible GSTs previously identified in tobacco and other dicotyledenous species. Recombinant Zm GST V-V showed high GST activity towards the diphenyl ether herbicide fluorodifen, detoxified toxic alkenal derivatives and reduced organic hydroperoxides. Antibodies raised to Zm GST I-II and Zm GST V-VI were used to monitor the expression of GST subunits in maize seedlings. Over a 24 h period the Zm GST I subunit was unresponsive to chemical treatment, while expression of Zm GST II was enhanced by auxins, herbicides, the herbicide safener dichlormid and glutathione. The Zm GST V subunit was more selective in its induction, only accumulating significantly in response to dichlormid treatment. During development Zm GST I and Zm GST V were expressed more in roots than in shoots, with Zm GST II expression limited to the roots.     

Plant glutathione S-transferases: enzymes with multiple functions in sickness and in health

Glutathione S-transferases (GSTs) are abundant proteins encoded by a highly divergent, ancient gene family. Soluble GSTs form dimers, each subunit of which contains active sites that bind glutathione and hydrophobic ligands. Plant GSTs attach glutathione to electrophilic xenobiotics, which tags them for vacuolar sequestration. The role of GSTs in metabolism is unclear, although their complex regulation by environmental stimuli implies that they have important protective functions. Recent studies show that GSTs catalyse glutathione-depend-ent isomerizations and the reduction of toxic organic hydroperoxides. GSTs might also have non-catalytic roles as carriers for phytochemicals.     

Screening for recombinant glutathione transferases active with monochlorobimane

A rapid and facile colony assay has been developed for catalytically active enzymes in combinatorial cDNA libraries of mutated glutathione transferases (GST), expressed in Escherichia coli. The basis of the method is the conjugation of glutathione (GSH) with the fluorogenic substrate monochlorobimane (MCB). This screening method makes it possible to isolate and characterize one recombinant clone that is active with MCB among thousands of inactive variants. Colonies containing GSTs that catalyze the conjugation of GSH with MCB display fluorescence under long-wavelength UV light. The fluorescence is visible instantly. One rat and 11 human GSTs representing four distinct enzyme classes were studied, and all except human GST T1-1 gave rise to fluorescent colonies. The colony assay based on MCB can consequently be broadly applied for identifying active GSTs both after subcloning of wild-type enzymes and in the screening of mutant libraries. Populations of bacteria expressing GSTs can also be analyzed by flow cytometry.     

Manipulation of plant tolerance to herbicides through co-ordinated metabolic engineering of a detoxifying glutathione transferase and thiol cosubstrate

The diphenyl ether herbicide fomesafen can be used selectively in soybean (Glycine max) due to its rapid detoxification by tau class glutathione transferases (GmGSTUs) which preferentially utilize the endogenous thiol homoglutathione (hGSH) as cosubstrate. Soybean cDNAs encoding GmGSTU21, which is highly active in detoxifying fomesafen, and an hGSH synthetase (GmhGS) have been cloned and functionally identified in Escherichia coli. Tobacco plants, which have limited GST activities towards fomesafen and which accumulate glutathione (GSH), rather than hGSH, have been transformed with either GmhGS alone, or a dual construct of GmhGS-GmGSTU21, both under the control of constitutive promoters. Using either construct, the transgenic tobacco accumulated hGSH, with a concomitant increase in GSH content. Segregating T1 plants were analysed for thiol content and GST activity towards fomesafen with GSH and hGSH as cosubstrates, and then scored for photobleaching injury caused by applications of fomesafen. These studies showed that hGSH accumulation alone gave no significant protection against the herbicide and that tolerance was only seen in plants which contained appreciable concentrations of hGSH and GmGSTU21 activity. Tolerance in the dual transformants was associated with the metabolism of radiolabelled fomesafen to inactive hGSH-derived conjugates, while susceptible lines were unable to detoxify the herbicide. These studies confirm the combined importance of specific GSTs and their preferred thiol cosubstrates in conferring herbicide selectivity traits in planta.     

A genomics approach to the comprehensive analysis of the glutathione S-transferase gene family in soybean and maize

By BLAST searching a large expressed sequence tag database for glutathione S-transferase (GST) sequences we have identified 25 soybean (Glycine max) and 42 maize (Zea mays) clones and obtained accurate full-length GST sequences. These clones probably represent the majority of members of the GST multigene family in these species. Plant GSTs are divided according to sequence similarity into three categories: types I, II, and III. Among these GSTs only the active site serine, as well as another serine and arginine in or near the "G-site" are conserved throughout. Type III GSTs have four conserved sequence patches mapping to distinct structural features. Expression analysis reveals the distribution of GSTs in different tissues and treatments: Maize GSTI is overall the most highly expressed in maize, whereas the previously unknown GmGST 8 is most abundant in soybean. Using DNA microarray analysis we observed increased expression among the type III GSTs after inducer treatment of maize shoots, with different genes responding to different treatments. Protein activity for a subset of GSTs varied widely with seven substrates, and any GST exhibiting greater than marginal activity with chloro-2,4 dinitrobenzene activity also exhibited significant activity with all other substrates, suggesting broad individual enzyme substrate specificity.     

Heme transport and detoxification in nematodes: subproteomics evidence of differential role of glutathione transferases

In contrast to their mammalian hosts, parasitic nematodes are heme auxotrophs and require pathways for the uptake and transport of exogenous heme for incorporation into hemoproteins. Phase II detoxification Nu-class glutathione transferase (GST) proteins have a proposed role as heme-binding ligandins in parasitic nematodes. The genome-verified free-living nematode Caenorhabditis elegans also cannot synthesize heme and is an ideal functional genomics model to delineate the role of individual nematode GSTs in heme trafficking and heme detoxification. In this study, C. elegans was exposed to externally controlled heme concentrations ranging from 20-fold suboptimal growth levels to 10-fold supra-optimal growth levels to mimic fluctuations in blood- and tissue-feeding parasitic cousins from the same nematode group. A new heme-responsive GST (GST-19) was identified by subproteomics approaches. Functional characterization of this and two other C. elegans GSTs revealed that they all have high affinity for heme compounds similar to mammalian soluble heme carrier proteins such as HBP23 ( K d approximately 10 (-8) M). In the genomics-predicted absence of orthologous mammalian soluble heme-binding proteins in nematodes, we propose that Nu-class GSTs are candidates in the cellular processing of heme compounds. Toxic heme binding may be coupled to enzymatic protection from its breakdown as several GSTs possess glutathione peroxidase activity.     

Proposed reductive metabolism of artemisinin by glutathione transferases in vitro

Artemisinin is a sesquiterpene lactone containing an endoperoxide bridge. It is a promising new antimalarial and is particularly useful against the drug resistant strains of Plasmodium falciparum. It has unique antimalarial properties since it acts through the generation of free radicals that alkylate parasite proteins. Since the antimalarial action of the drug is antagonised by glutathione and ascorbate and has unusual pharmacokinetic properties in humans, we have investigated if the drug is broken down by a typical reductive reaction in the presence of glutathione transferases. Cytosolic glutathione transferases (GSTs) detoxify electrophilic xenobiotics by catalysing the formation of glutathione (GSH) conjugates and exhibit glutathione peroxidase activity towards hydroperoxides. Artemisinin was incubated with glutathione, NADPH and glutathione reductase and GSTs in a coupled assay system analogous to the standard assay scheme with cumene hydroperoxide as a substrate of GSTs. Artemisinin was shown to stimulate NADPH oxidation in cytosols from rat liver, kidney, intestines and in affinity purified preparations of GSTs from rat liver. Using human recombinant GSTs hetelorogously expressed in Escherichia coli, artemisinin was similarly shown to stimulate NADPH oxidation with the highest activity observed with GST M1-1. Using recombinant GSTs the activity of GSTs with artemisinin was at least two fold higher than the reaction with CDNB. Considering these results, it is possible that GSTs may contribute to the metabolism of artemisinin in the presence of NADPH and GSSG-reductase We propose a model, based on the known reactions of GSTs and sesquiterpenes, in which (1) artemisinin reacts with GSH resulting in oxidised glutathione; (2) the oxidised glutathione is then converted to reduced glutathione via glutathione reductase; and (3) the latter reaction may then result in the depletion of NADPH via GSSG-reductase. The ability of artemisinin to react with GSH in the presence of GST may be responsible for the NADPH utilisation observed in vitro and suggests that cytosolic GSTs are likely to be contributing to metabolism of artemisinin and related drugs in vivo.     

Forced evolution of a herbicide detoxifying glutathione transferase

Plant Tau class glutathione transferases (GSTUs) detoxify diphenylether herbicides such as fluorodifen, determining their selectivity in crops and weeds. Using reconstructive PCR, a series of mutant GSTUs were generated from in vitro recombination and mutagenesis of the maize sequences ZmGSTU1 and ZmGSTU2 (with the prefix Zm designating Zea mays L.). A screen of 5000 mutant GSTUs identified seven enzymes with enhanced fluorodifen detoxifying activity. The best performing enhanced fluorodifen detoxifying mutant (EFD) had activity 19-fold higher than the parent enzymes, with a single point mutation conferring this enhancement. Further mutagenesis of this residue generated an EFD with a 29-fold higher catalytic efficiency toward fluorodifen as compared with the parents but with unaltered catalysis toward other substrates. When expressed in Arabidopsis thaliana, the optimized EFD, but not the parent enzymes, conferred enhanced tolerance to fluorodifen. Molecular modeling predicts that the serendipitous mutation giving the improvement in detoxification is due to the removal of an unfavorable interaction together with the introduction of a favorable change in conformation of residues 107-119, which contribute to herbicide binding.     

Molecular cloning and characterization of three sigma glutathione S-transferases from disk abalone (Haliotis discus discus)

Three novel glutathione S-transferase (GSTs) cDNAs were cloned from a disk abalone (Haliotis dicus discus) cDNA library. Multiple alignment and phylogenetic analysis of three GSTs revealed that their closest relationship is with insect sigma GSTs. Recombinant GSTs were over-expressed in Escherichia coli as soluble fusion proteins. HdGSTS1 and HdGSTS2 were active towards 1-chloro-2,4-dinitrobenzene and ethacrynic acid, whereas HdGSTS3 appeared to be a non-enzymatic GST. Two active GSTs had similar optimum conditions for enzymatic reaction at pH 8.0 and temperature of approximately 30 degrees C. Molecular modeling analysis of three GSTs implicates their diverse active sites as being responsible for their different enzymatic features. Three sigma GSTs had significantly different expression patterns and levels of expression in abalone tissues, indicating their different functions. After 48 h-exposure to three model marine pollutants, only HdGSTS1 exhibited a proper inducibility, exhibiting its good biomarker potential for organic contaminants in marine environment. In contrast, the other two sigma GSTs revealed a minor role in the response of pollutants exposure.     

Binding and protection of porphyrins by glutathione S-transferases of Zea mays L.

Glutathione S-transferases (GSTs) are multi-functional enzymes, known to conjugate xenobiotics and degrade peroxides. Herein, we report on the potential of four Zea mays GST isoforms (Zm GST I–I, Zm GST I–II, Zm GST II–II and Zm GST III–III) to act as binding and protection proteins. These isoforms bind protoporphyrin IX (PPIX), mesoporphyrin, coproporphyrin, uroporphyrin and Mg-protoporpyhrin, but do not form a glutathione conjugate. The binding is non-covalent and inhibits GSTs enzymatic activity, dependent on the type of the porphyrin and GST isoform tested. I₅₀ values are in the range of 1 to 10 μM for PPIX, the inhibition by mesoporphyrin and Mg-protoporphyrin (Mg-PPIX) is two to five times less. The mode of binding is non-competitive for the hydrophobic substrate and competitive for glutathione. Binding affinities (K_D values) of the GST isoforms are between 0.3 and 0.8 μM for coproporphyrin and about 2 μM for mesoporphyrin.Zm GST III–III prevents the nonenzymatic autoxidation of protoporphyrinogen to the phytotoxic PPIX. Zm GST II–II can reduce the oxidative degradation of hemin. This points to a specific ligand role of distinct GST isoforms to protect tetrapyrroles in the plant cell.     

Partial characterization of glutathione S-transferases from wheat (Triticum spp.) and purification of a safener-induced glutathione S-transferase from Triticum tauschii.

Hexaploid wheat (Triticum aestivum L.) has very low constitutive glutathione S-transferase (GST) activity when assayed with the chloroacetamide herbicide dimethenamid as a substrate, which may account for its low tolerance to dimethenamid in the field. Treatment of seeds with the herbicide safener fluxofenim increased the total GST activity extracted from T. aestivum shoots 9-fold when assayed with dimethenamid as a substrate, but had no effect on glutathione levels. Total GST activity in crude protein extracts from T. aestivum, Triticum durum, and Triticum tauschii was separated into several component GST activities by anion-exchange fast-protein liquid chromatography. These activities (isozymes) differed with respect to their activities toward dimethenamid or 1-chloro-2,4-dinitrobenzene as substrates and in their levels of induction by safener treatment. A safener-induced GST isozyme was subsequently purified by anion-exchange and affinity chromatography from etiolated shoots of the diploid wheat species T. tauschii (a progenitor of hexaploid wheat) treated with the herbicide safener cloquintocet-mexyl. The isozyme bound to a dimethenamid-affinity column and had a subunit molecular mass of 26 kD based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme (designated GST TSI-1) was recognized by an antiserum raised against a mixture of maize (Zea mays) GSTs. Amino acid sequences obtained from protease-digested GST TSI-1 had significant homology with the safener-inducible maize GST V and two auxin-regulated tobacco (Nicotiana tabacum) GST isozymes.     

Whole genome survey of the glutathione transferase family in the brown algal model Ectocarpus siliculosus

We report here an exhaustive analysis of the glutathione transferases (GSTs) in the model brown alga Ectocarpus siliculosus using available genomic resources. A genome survey revealed the presence of twelve cytosolic GSTs, belonging to the Sigma class, two pseudogenes, one GST of the Kappa class, and three microsomal GSTs of the MGST3 family of membrane associated protein involved in eicosanoid and glutathione metabolism. Gene structure and phylogenetic analyses demonstrated the partition of the Sigma GSTs into two clusters which have probably evolved by duplication events. Gene expression profiling was conducted after the addition of high concentrations of chemicals, such as H(2)O(2), herbicides, heavy metals, as well as fatty acid derivatives, in order to induce stress conditions and to monitor early response mechanisms. The results of these experiments suggested that E. siliculosus GST genes are recruited in different and specific conditions. In addition, heterologous expression in yeast of two E. siliculosus microsomal GST showed that these enzymes feature peroxidase rather than transferase activity. The potential involvement of E. siliculosus GST in the metabolism of oxygenated polyunsaturated fatty acids is discussed.     

Plant glutathione transferases

The soluble glutathione transferases (GSTs, EC 2.5.1.18) are encoded by a large and diverse gene family in plants, which can be divided on the basis of sequence identity into the phi, tau, theta, zeta and lambda classes. The theta and zeta GSTs have counterparts in animals but the other classes are plant-specific and form the focus of this article. The genome of Arabidopsis thaliana contains 48 GST genes, with the tau and phi classes being the most numerous. The GST proteins have evolved by gene duplication to perform a range of functional roles using the tripeptide glutathione (GSH) as a cosubstrate or coenzyme. GSTs are predominantly expressed in the cytosol, where their GSH-dependent catalytic functions include the conjugation and resulting detoxification of herbicides, the reduction of organic hydroperoxides formed during oxidative stress and the isomerization of maleylacetoacetate to fumarylacetoacetate, a key step in the catabolism of tyrosine. GSTs also have non-catalytic roles, binding flavonoid natural products in the cytosol prior to their deposition in the vacuole. Recent studies have also implicated GSTs as components of ultraviolet-inducible cell signaling pathways and as potential regulators of apoptosis. Although sequence diversification has produced GSTs with multiple functions, the structure of these proteins has been highly conserved. The GSTs thus represent an excellent example of how protein families can diversify to fulfill multiple functions while conserving form and structure.     

Intrinsic electrophilicity of the 4-methylsulfonyl-2-pyridone scaffold in glucokinase activators: Role of glutathione-S-transferases and in vivo quantitation of a glutathione conjugate in rats

Previous studies on the in vitro metabolism of 4-alkylsulfonyl-2-pyridone-based glucokinase activators revealed a facile, non-enzymatic displacement of the 4-alkylsulfonyl group by glutathione. In the present studies, a role for glutathione-S-transferases (GST) as catalysts in the desulfonylation reaction was demonstrated using a combination of human liver microsomes, human liver cytosol and human GSTs. The identification of a glutathione conjugate in circulation following intravenous administration of a candidate 4-methylsulfonyl-2-pyridone to rats confirmed the relevance of the in vitro findings.