GST profile expression study in some selected plants: in silico approach

Glutathione acts as a protein disulfide reductant, which detoxifies herbicides by conjugation, either spontaneously or by the activity of one of a number of glutathione-S-transferases (GSTs), and regulates gene expression in response to environmental stress and pathogen attack. GSTs play role in both normal cellular metabolism as well as in the detoxification of a wide variety of xenobiotic compounds, and they have been intensively studied with regard to herbicide detoxification in plants. A newly discovered plant GST subclass has been implicated in numerous stress responses, including those arising from pathogen attack, oxidative stress, and heavy-metal toxicity. In addition, plant GSTs play a role in the cellular response to auxins and during the normal metabolism of plant secondary products like anthocyanins and cinnamic acid. The present study involves two in silico analytical approaches—general secondary structure prediction studies of the proteins and detailed signature pattern studies of some selected GST classes in Arabdiopsis thaliana, Mustard, Maize, and Bread wheat by standard Bioinformatics tools; structure prediction tools; signature pattern tools; and the evolutionary trends were analyzed by ClustalW. For this purpose, sequences were obtained from standard databases. The study reveals that these proteins are mainly alpha helical in nature with specific signature pattern similar to phosphokinase C, tyrosine kinase, and casein kinase II proteins, which are closely related to plant oxidative stress. This study aims to comprehend the relationship of GST gene family and plant oxidative stress with respect to certain specific conserved motifs, which may help in future studies for screening of biomodulators involved in plant stress metabolism.     

GST profile expression study in some selected plants: in silico approach

Glutathione acts as a protein disulphide reductant, which detoxifies herbicides by conjugation, either spontaneously or by the activity of one of a number of glutathione-S-transferases (GSTs), and regulates gene expression in response to environmental stress and pathogen attack. GSTs play roles in both normal cellular metabolisms as well as in the detoxification of a wide variety of xenobiotic compounds, and they have been intensively studied with regard to herbicide detoxification in plants. A newly discovered plant GST subclass has been implicated in numerous stress responses, including those arising from pathogen attack, oxidative stress and heavy-metal toxicity. In addition, plants GSTs play a role in the cellular response to auxins and during the normal metabolism of plant secondary products like anthocyanins and cinnamic acid. The present work involves two in silico analytical approaches—general secondary structure prediction studies of the proteins and detailed signature pattern studies of some selected GST classes in Arabdiopsis thaliana, mustard, maize and bread wheat by standard Bioinformatics tools; structure prediction tools; signature pattern tools; and the evolutionary trends were analyzed by ClustalW. For this purpose, sequences were obtained from standard databases. The work reveals that these proteins are mainly alpha helical in nature with specific signature pattern similar to phosphokinase C, tyrosine kinase and casein kinase II proteins, which are closely related to plant oxidative stress. This study aims to comprehend the relationship of GST gene family and plant oxidative stress with respect to certain specific conserved motifs, which may help in future studies for screening of biomodulators involved in plant stress metabolism.     

The plant glutathione transferase gene family: genomic structure, functions, expression and evolution

Glutathione transferases (GSTs) are ubiquitous, multifunctional proteins encoded by large gene families. In different plant species this gene family is comprised of 25–60 members, that can be grouped into six classes on the basis of sequence identity, gene organization and active site residues in the protein. The Phi and Tau classes are the most represented and are plant specific, while Zeta and Theta GSTs are found also in animals. Despite pronounced sequence and functional diversification, GSTs have maintained a highly conserved three-dimensional structure through evolution. Most GSTs are cytosolic and active as dimers, performing diverse catalytic as well as non-catalytic roles in detoxification of xenobiotics, prevention of oxidative damage and endogenous metabolism. Among their catalytic activities are the conjugation of electrophilic substrates to glutathione, glutathione-dependent isomerizations and reductions of toxic organic hydroperoxides. Their main non-catalytic role is as hormone and flavonoid ligandins. GST genes are predominantly organized in clusters non-randomly distributed in the genome. Phylogenetic studies indicate that plant GSTs have mainly evolved after the divergence of plants, the two prevalent Phi and Tau classes being the result of recent, multiple duplication events.     

Characterisation of a zeta class glutathione transferase from Arabidopsis thaliana with a putative role in tyrosine catabolism

A glutathione transferase (GST) similar to zeta GSTs in animals and fungi has been cloned from Arabidopsis thaliana using RT-PCR. The Arabidopsis zeta GST (AtGSTZ1) was expressed in Escherichia coli as his-tagged polypeptides, which associated together to form the 50-kDa AtGSTZ1-1 homodimer. Following purification, AtGSTZ1-1 was assayed for a range of activities and compared with other purified recombinant plant GSTs from the phi, tau, and theta classes. AtGSTZ1-1 differed from the other GSTs in showing no glutathione conjugating activity toward xenobiotics and no glutathione peroxidase activity toward organic hydroperoxides. Uniquely among the plant GSTs, AtGSTZ1-1 showed activity as a maleylacetone isomerase (MAI). This glutathione-dependent reaction is analogous to the cis-trans isomerization of maleylacetoacetate to fumarylacetoacetate, which occurs in the course of tyrosine catabolism to acetoacetate and fumarate. Thus, rather than functioning as a conventional GST, AtGSTZ1-1 appears to be involved in tyrosine degradation. In addition to the MAI activity, the AtGSTZ1-1 also catalyzed the glutathione-dependent dehalogenation of dichloroacetic acid to glyoxylic acid. This latter activity was used to demonstrate the presence of functional AtGSTZ1-1 inplanta.     

昆虫谷胱甘肽S-转移酶的基因结构及其表达调控

谷胱甘肽S-转移酶（glutathione S-transferases, GSTs）属于一个超家族,目前已从20多种昆虫中克隆得到了近百个GSTs基因序列。这些基因分属于至少3个类别,Ⅰ（Delta）类,Ⅱ类和Ⅲ（Epsilon）类,其中Ⅰ类和Ⅲ类是昆虫特异性的类别。昆虫Ⅰ类GSTs基因通常由多基因家族编码,基因多态性在不同昆虫种类中差异很大。Ⅱ类基因的种类较少,基因的结构较简单,通常是单拷贝基因。Ⅲ类基因是最近才鉴定出来的新类别,目前仅在黑腹果蝇和冈比亚按蚊中明确了其在染色体上的定位。基因簇、可变剪接和基因融合等机制是导致昆虫GSTs基因多态性的主要原因。在抗性昆虫种群中,GSTs表达量的增加有mRNA水平的提高和基因扩增两种机制,但后一种机制的报道很少。GSTs活性的增加是由于属于一类或多类的多个同工酶的增量调控,也有少数是由于单个同工酶的增量调控。GSTs的表达受反式调控元件和顺式调控元件的调控。目前仅有少数含有调节基因的染色体大致位点和可能的调控元件得到鉴定。     

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.     

Primary sequence heterogeneity and tissue expression of glutathione S-transferases of Fasciola hepatica.

Glutathione S-transferases (GSTs) from Fasciola hepatica have been purified by glutathione affinity chromatography. Two closely migrating species of Mr 26,000 and 26,500 were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and several species resolved by two-dimensional gel analysis, indicating substantial heterogeneity among the GSTs. N-terminal amino acid sequencing revealed one core sequence containing three polymorphisms, whereas the sequence of GST peptides implied a minimum of three different GSTs. The amino acid sequence data assigned the F. hepatica GSTs to the mu class of GSTs with high similarities to these proteins in other helminths and mammals. The native GSTs of F. hepatica appeared to behave as dimers as determined by molecular sieving chromatography. The observation that the GSTs of F. hepatica are heterogeneous in sequence and behave as dimers in the native state suggest that these isoenzymes may exhibit considerable functional heterogeneity which may be of importance to the parasite. Immunocytochemical studies suggest that the main source of GST in F. hepatica are the parenchymal cells and peripheral tissues of the parasite. Some extracellular GST is associated with the lamellae of the intestinal epithelium. The identification of an intestinal GST is unique among trematodes studied to date.     

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.     

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.     

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.     

Novel immunochemical characteristics of glutathione S-transferase isozymes of rat liver cytosol

Reevaluation of the immunochemical relationships among the individual glutathione S-transferase (GST) isozymes (GSTs 1-1, 1-2, 2-2, 3-3, 3-4, 4-4, and GSTs with isoelectric points of 7.5 and 6.8) of rat liver cytosol was performed utilizing the immunoblot technique. As a result, we found that the respective isozymes of two isozyme classes of rat liver cytosol might possess a common epitope(s) which has been undetected by the Ouchterlony double-diffusion method. The assumption was further supported by the results of the effects of Fab' prepared from some anti-GST antibodies on the enzymatic activity of GSTs.     

Characterization of glutathione S-transferases in rat kidney. Alteration of composition by cis-platinum.

We have developed chromatographic and mathematical protocols that allowed the high resolution of glutathione S-transferase (GST) subunits, and the identification of a previously unresolved GST monomer in rat kidney cytosol; the monomer was identified tentatively as subunit 6. Also, an aberrant form of GST 7-7 dimer appeared to be present in the kidney. This development was utilized to illustrate the response of rat kidney GST following cis-platinum treatment in vivo. Rat kidney cytosol was separated into three 'affinity families' of GST activity after elution from a GSH-agarose matrix. The affinity peaks were characterized by quantitative differences in their subunit and dimeric compositions as determined by subsequent chromatography on a cation-exchange matrix and specific activity towards substrates. By use of these criteria, the major GST dimers of affinity peaks were tentatively identified. The major GST dimers in peak I were GST 1-1 and 1-2, in affinity peak II it was GST 2-2, and in peak III they were GST 3-3 and 7-7. GST 3-6 and/or 4-6, which have not been previously resolved in kidney cytosol, were also present in peak II. Alterations in the kidney cytosolic GST composition of male rats were detected subsequent to the administration of cis-platinum (7.0 mg/kg subcutaneously, 6 days). This treatment caused a pronounced alteration in the GST profile, and the pattern of alteration was markedly different from that reported for other chemicals in the kidney or in the liver. In general, the cellular contents of the GSTs of the Alpha and the Mu classes decreased and increased respectively. It is postulated that the decrease in the Alpha class of GSTs by cis-platinum treatment may be related to renal cortical damage and the loss of GSTs in the urine. The increase in the Mu class of GSTs could potentially stem from a lowered serum concentration of testosterone; the latter is a known effect of cis-platinum treatment.     

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".     

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.     

A novel glutathione transferase from Haemophilus influenzae which has high affinity towards antibiotics

Cytosolic glutathione transferase (GSTs) are a family of multi-functional proteins which catalyse the conjugation of glutathione (GSH) to a large variety of endogenous and exogenous electrophilic compounds. Much is known about cytosolic mammalian GSTs, however, the presence of GSTs in several aerobic and anaerobic micro-organisms has also been demonstrated. Several findings seem to suggest that bacterial GSTs are involved in processes of biodegradation of xenobiotics, including antibiotics. However, the function played by these enzymes in the bacterial cell still remains to be clarified. At present, it is ill-defined whether bacterial GST can be classified, as in the case of mammalian enzymes, into several distinct classes.Here we report the purification of a GST isoform from Haemophilus influenzae using GSH-affinity chromatography. The purified protein was characterised by immunological and kinetic properties different from other known GSTs. The dissociation constants of chloramphenicol, ampicillin, rifampicin and tetracycline to the purified enzyme were 0.62, 9.06, 4.08 and 1.77 microM, respectively, as determined by following the quenching of the protein intrinsic fluorescence. These values were much lower than those previously determined for the same drugs with other mammalian or bacterial GSTs.The present results indicate that the enzyme purified from H. influenzae is a novel GST isoform well distinguished from other known mammalian or bacterial GSTs.     

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.     

Characterization of the activity and folding of the glutathione transferase from Escherichia coli and the roles of residues Cys(10) and His(106)

GSTs (glutathione transferases) are an important class of enzymes involved in cellular detoxification. GSTs are found in all classes of organisms and are implicated in resistance towards drugs, pesticides, herbicides and antibiotics. The activity, structure and folding, particularly of eukaryotic GSTs, have therefore been widely studied. The crystal structure of EGST (GST from Escherichia coli) was reported around 10 years ago and it suggested Cys(10) and His(106) as potential catalytic residues. However, the role of these residues in catalysis has not been further investigated, nor have the folding properties of the protein been described. In the present study we investigated the contributions of residues Cys(10) and His(106) to the activity and stability of EGST. We found that EGST shows a complex equilibrium unfolding profile, involving a population of at least two partially folded intermediates, one of which is dimeric. Mutation of residues Cys(10) and His(106) leads to stabilization of the protein and affects the apparent steady-state kinetic parameters for enzyme catalysis. The results suggest that the imidazole ring of His(106) plays an important role in the catalytic mechanism of the enzyme, whereas Cys(10) is involved in binding of the substrate, glutathione. Engineering of the Cys(10) site can be used to increase both the stability and GST activity of EGST. However, in addition to GST activity, we discovered that EGST also possesses thiol:disulfide oxidoreductase activity, for which the residue Cys(10) plays an essential role. Further, tryptophan quenching experiments indicate that a mixed disulfide is formed between the free thiol group of Cys(10) and the substrate, glutathione.