A Tale of Plant Glutathione <Emphasis Type="Italic">S</Emphasis>-Transferases: Since 1970

Ubiquitously distributed multifunctional superfamily of Glutathione S-transferases (GST) generally constitute a dimeric enzymes and catalyse the conjugation of the thiol group of the glutathione (GSH) to diverse electrophilic centres on lipophilic molecules with the formation of rather less active end products. Besides their well investigated conjugation reaction for the detoxification of endogenous and xenobiotic compounds, they can also be involved in both GSH dependent peroxidation or isomerization reactions, and several other non-catalytic functions, like binding of non-substrate ligands, stress-induced signalling processes and preventing of apoptosis. Plant GSTs have been a focus of attention because of their roles in herbicide detoxification and today seven distinct classes of soluble (cytosolic) GSTs are presented as Phi, Tau, Theta, Zeta, Lambda, Dehydroascorbate reductases (DHARs) and Tetrachlorohydroquinone dehalogenase (TCHQD). While GSTs show overall sequence diversification within and between classes, they retain a high level of three-dimensional structure conservation over long evolutionary periods. In this review mainly the soluble plant GSTs will be considered by giving attention to their structures, subcellular localizations, genomic organizations, catalytic/noncatalytic functions, and comparisons given with respect to their mammalian counterparts where necessary.     

Organisation and structural evolution of the rice glutathione <Emphasis Type="Italic">S</Emphasis>-transferase gene family

Glutathione S-transferases (GSTs) comprise a large family of key defence enzymes against xenobiotic toxicity. Here we describe the comprehensive characterisation of this important multigene family in the model monocot species rice [Oryza sativa (L.)]. Furthermore, we investigate the molecular evolution of the family based on the analysis of (1) the patterns of within-genome duplication, and (2) the phylogenetic relationships and evolutionary divergence among rice, Arabidopsis, maize and soybean GSTs. By in-silico screening of the EST and genome divisions of the Genbank/EMBL/DDBJ database we have isolated 59 putative genes and two pseudogenes, making this the largest plant GST family characterised to date. Of these, 38 (62%) are represented by genomic and EST sequences and 23 (38%) are known only from their genomic sequences. A preliminary survey of EST collections shows a large degree of variability in gene expression between different tissues and environmental conditions, with a small number of genes (13) accounting for 80% of all ESTs. Rice GSTs are organised in four main phylogenetic classes, with 91% of all rice genes belonging to the two plant-specific classes Tau (40 genes) and Phi (16 genes). Pairwise identity scores range between 17 and 98% for proteins of the same class, and 7 and 21% for interclass comparisons. Rapid evolution by gene duplication is suggested by the discovery of two large clusters of 7 and 23 closely related genes on chromosomes 1 and 10, respectively. A comparison of the complete GST families in two monocot and two dicot species suggests a monophyletic origin for all Theta and Zeta GSTs, and no more than three common ancestors for all Phi and Tau genes.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by M.-A. Grandbastien     

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.     

Divergence in structure and function of tau class glutathione transferase from Pinus tabulaeformis,P. yunnanensis and P. densata

Glutathione transferases (GSTs) are a family of enzymes that play important roles in stress tolerance and detoxification in plants. The plant GSTs are divided into four classes (phi, tau, zeta and theta), among which tau is the most numerously represented. To date, studies on GSTs in plants have focused largely on crop species. There is extremely little information on the molecular characteristics of GSTs in gymnosperms. Generalization on GST characteristics unique to gymnosperms and the patterns of GST evolution in plants cannot be made before more members of the gene family in conifers are described. In this study we report three new GSTs from Pinus tabulaeformis, Pinus densata and Pinus yunnanensis. Structural and phylogenetic analyses placed these three GSTs in tau class. The tau GST class is subdivided into three clades and this subdivision seems an ancient event that may have pre-dated the gymnosperm and angiosperm split. Sequence analysis revealed a highly conserved N-terminal domain in contrast to a highly variable C-terminal domain. Mutations even outside the critical glutathione-binding site in N-terminal domain can have pronounced effect on GST catalytic property. Thus, sequence similarity does not parallel functional specificity. The high diversity in C-terminal domain determines a wide range of substrate selectivity and specificity among tau GSTs. Thus the a few conserved residues in C-terminal domain seem essential to maintain the structure of the domain and the protein dimer. More extensive data on GST family organization and a thorough gene-by-gene analysis in conifers are needed to advance our understanding of the true diversity and evolution of GST in structure and function in plants.     

Glutathione transferase-like proteins encoded in genomes of yeasts and fungi: insights into evolution of a multifunctional protein superfamily

Most fungal glutathione transferases (GSTs) do not fit easily into any of the previously characterised classes by immunological, sequence or catalytic criteria. In contrast to the paucity of studies on GSTs cloned or isolated from fungal sources, a screen of databases revealed 67 GST-like sequences from 21 fungal species. Comparison by multiple sequence alignment generated a dendrogram revealing five clusters of GST-like proteins designated clusters 1, 2, EFIBgamma, Ure2p and MAK16, the last three of which have previously been related to the GST superfamily. Surprisingly, a relatively small number of fungal GSTs belong to mainstream classes and the previously-described fungal Gamma class is not widespread in the 21 species studied. Representative crystal structures are available for the EFIBgamma and Ure2p classes and the domain structures of representative sequences are compared with these. In addition, there are some "orphan" sequences that do not fit into any previously-described class, but show similarity to genes implicated in fungal biosynthetic gene clusters. We suggest that GST-like sequences are widespread in fungi, participating in a wide range of functions. They probably evolved by a process similar to domain "shuffling".     

Differences in the subunit interface residues of alternatively spliced glutathione transferases affects catalytic and structural functions

GSTs (glutathione transferases) are multifunctional widespread enzymes. Currently there are 13 identified classes within this family. Previously most structural characterization has been reported for mammalian Alpha, Mu and Pi class GSTs. In the present study we characterize two enzymes from the insect-specific Delta class, adGSTD3-3 and adGSTD4-4. These two proteins are alternatively spliced products from the same gene and have very similar tertiary structures. Several major contributions to the dimer interface area can be separated into three regions: conserved electrostatic interactions in region 1, hydrophobic interactions in region 2 and an ionic network in region 3. The four amino acid side chains studied in region 1 interact with each other as a planar rectangle. These interactions are highly conserved among the GST classes, Delta, Sigma and Theta. The hydrophobic residues in region 2 are not only subunit interface residues but also active site residues. Overall these three regions provide important contributions to stabilization and folding of the protein. In addition, decreases in yield as well as catalytic activity changes, suggest that the mutations in these regions can disrupt the active site conformation which decreases binding affinity, alters kinetic constants and alters substrate specificity. Several of these residues have only a slight effect on the initial folding of each subunit but have more influence on the dimerization process as well as impacting upon appropriate active site conformation. The results also suggest that even splicing products from the same gene may have specific features in the subunit interface area that would preclude heterodimerization.     

Characterization of two Arabidopsis thaliana glutathione S-transferases

Glutathione S-transferases (GST) are multifunctional proteins encoded by a large gene family, divided on the basis of sequence identity into phi, tau, theta, zeta and lambda classes. The phi and tau classes are present only in plants. GSTs appear to be ubiquitous in plants and are involved in herbicide detoxification and stress response, but little is known about the precise role of GSTs in normal plant physiology and during biotic and abiotic stress response. Two cDNAs representing the two plant classes tau and phi, AtGSTF9 and AtGSTU26, were expressed in vitro and the corresponding proteins were analysed. Both GSTs were able to catalyse a glutathione conjugation to 1-chloro-2,4-dinitrobenzene (CDNB), but they were inactive as transferases towards p-nitrobenzylchloride (pNBC). AtGSTF9 showed activity towards benzyl isothiocyanate (BITC) and an activity as glutathione peroxidase with cumene hydroperoxide (CumHPO). AtGSTU26 was not active as glutathione peroxidase and towards BITC. RT-PCR analysis was used to evaluate the expression of the two genes in response to treatment with herbicides and safeners, chemicals, low and high temperature. Our results reveal that AtGSTU26 is induced by the chloroacetanilide herbicides alachlor and metolachlor and the safener benoxacor, and after exposure to low temperatures. In contrast, AtGSTF9 seems not to be influenced by the treatments employed.     

Structural and Functional Evolution of Positively Selected Sites in Pine Glutathione S-Transferase Enzyme Family

Phylogenetic analyses have identified positive selection as an important driver of protein evolution, both structural and functional. However, the lack of appropriate combined functional and structural assays has generally hindered attempts to elucidate patterns of positively selected sites and their effects on enzyme activity and substrate specificity. In this study we investigated the evolutionary divergence of the glutathione S-transferase (GST) family in Pinus tabuliformis, a pine that is widely distributed from northern to central China, including cold temperate and drought-stressed regions. GSTs play important roles in plant stress tolerance and detoxification. We cloned 44 GST genes from P. tabuliformis and found that 26 of the 44 belong to the largest (Tau) class of GSTs and are differentially expressed across tissues and developmental stages. Substitution models identified five positively selected sites in the Tau GSTs. To examine the functional significance of these positively selected sites, we applied protein structural modeling and site-directed mutagenesis. We found that four of the five positively selected sites significantly affect the enzyme activity and specificity; thus their variation broadens the GST family substrate spectrum. In addition, positive selection has mainly acted on secondary substrate binding sites or sites close to (but not directly at) the primary substrate binding site; thus their variation enables the acquisition of new catalytic functions without compromising the protein primary biochemical properties. Our study sheds light on selective aspects of the functional and structural divergence of the GST family in pine and other organisms.     

Expression and characterization of a novel class of glutathione S-transferase from Anopheles dirus

A new Anopheles dirus glutathione S-transferase (GST) has been obtained and named adGST4-1. Both genomic DNA and cDNA for heterologous expression were acquired. The genomic sequence was 3188bp and consisted of the GST gene as well as flanking sequence. The flanking sequence was analyzed for possible regulatory elements that would control gene expression. In Drosophila several of these elements have been shown to be involved in development and cell differentiation. The deduced amino acid sequence has low identity compared with the four alternatively spliced enzymes, adGST1-1 to 1-4, from another An. dirus GST gene adgst1AS1. The percent identities are 30--40% and 11--12% comparing adGST4-1 to insect GSTs from Delta and Sigma classes, respectively. Enzyme characterization of adGST4-1 shows it to be distinct from the other An. dirus GSTs because of low enzyme activity for customary GST substrates including 1-chloro-2, 4-dinitrobenzene (CDNB). However, this enzyme has a greater affinity of interaction with pyrethroids compared to the other An. dirus GSTs.     

Novel class of glutathione transferases from cyanobacteria exhibit high catalytic activities towards naturally occurring isothiocyanates

In the present paper, we report a novel class of GSTs (glutathione transferases), called the Chi class, originating from cyanobacteria and with properties not observed previously in prokaryotic enzymes. GSTs constitute a widespread multifunctional group of proteins, of which mammalian enzymes are the best characterized. Although GSTs have their origin in prokaryotes, few bacterial representatives have been characterized in detail, and the catalytic activities and substrate specificities observed have generally been very modest. The few well-studied bacterial GSTs have largely unknown physiological functions. Genome databases reveal that cyanobacteria have an extensive arsenal of glutathione-associated proteins. We have studied two cyanobacterial GSTs which are the first examples of bacterial enzymes that are as catalytically efficient as the best mammalian enzymes. GSTs from the thermophile Thermosynechococcus elongatus BP-1 and from Synechococcus elongatus PCC 6301 were found to catalyse the conjugation of naturally occurring plant-derived isothiocyanates to glutathione at high rates. The cyanobacterial GSTs studied are smaller than previously described members of this enzyme family, but display many of the typical structural features that are characteristics of GSTs. They are also active towards several classical substrates, but at the same moderate rates that have been observed for other GSTs derived from prokaryotes. The cloning, expression and characterization of two cyanobacterial GSTs are described. The possible significance of the observed catalytic properties is discussed in the context of physiological relevance and GST evolution.     

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.     

Catalytic properties of glutathione-binding residues in a tau class glutathione transferase (PtGSTU1) from Pinus tabulaeformis

Glutathione transferases (GSTs) play important roles in stress tolerance and detoxification in plants. However, there is extremely little information on the molecular characteristics of GSTs in gymnosperms. In a previous study, we cloned a tau class GST (PtGSTU1) from a gymnosperm (Pinus tabulaeformis) for the first time. Based on the N-terminal amino acid sequence identity to the available crystal structures of plant tau GSTs, Ser13, Lys40, Ile54, Glu66 and Ser67 of PtGSTU1 were proposed as glutathione-binding (G-site) residues. The importance of Ser13 as a G-site residue was investigated previously. The functions of Lys40, Ile54, Glu66 and Ser67 of PtGSTU1 are examined in this study through site-directed mutagenesis. Enzyme assays and thermal stability measurements on the purified recombinant PtGSTU1 showed that substitution at each of these sites significantly affects the enzyme's substrate specificity and affinity for GSH, and these residues are essential for maintaining the stability of PtGSTU1. The results of protein expression and refolding analyses suggest that Ile54 is involved in the protein folding process. The findings demonstrate that the aforementioned residues are critical components of active sites that contribute to the enzyme's catalytic activity and structural stability.     

Organ-specific expression of glutathione S-transferases and the efficacy of herbicide safeners in Arabidopsis

The functions of plant glutathione S-transferases (GSTs) under normal growth conditions are poorly understood, but their activity as detoxification enzymes has been harnessed in agriculture for selective weed control. Herbicide safeners protect monocot crops from herbicide injury but have little effect on weedy monocot or dicot species. Protection by safeners is associated with expression of herbicide-metabolizing enzymes including GSTs, but the basis for selective action of safeners between monocots and dicots is not known. To address this question we have studied the response of Arabidopsis (Arabidopsis thaliana) to various safeners. Benoxacor, fenclorim, and fluxofenim did not protect Arabidopsis from herbicide injury but did induce RNA expression of the glutathione-conjugate transporters encoded by AtMRP1, AtMRP2, AtMRP3, and AtMRP4. These safeners also induced the organ-specific expression of AtGSTU19 and AtGSTF2, two previously characterized Arabidopsis GSTs from different classes of this enzyme family. RNA hybridization, immunoblot, and reporter gene analyses indicated expression of AtGSTU19 induced by safeners predominated in roots. To test the hypothesis that increased expression of AtGSTU19 would be sufficient to provide tolerance to chloroacetamide herbicides, a chimeric gene was produced containing the open reading frame for this GST driven by a constitutive promoter. Plants containing this transgene had a modest increase in AtGSTU19 protein, predominantly in roots, but this had no effect on tolerance to chloroacetamide herbicides. The localized induction of GSTs by safeners in roots of Arabidopsis may explain why these compounds are unable to provide herbicide tolerance to dicot plant species.