Genome-wide identification and expression profiling of glutathione S-transferase family under multiple abiotic and biotic stresses in Medicago truncatula L.

Glutathione transferases (GSTs) constitute an ancient, ubiquitous, multi-functional antioxidant enzyme superfamily that has great importance on cellular detoxification against abiotic and biotic stresses as well as plant development and growth. The present study aimed to a comprehensive genome-wide identification and functional characterization of GST family in one of the economically important legume plants-Medicago truncatula. Here, we have identified a total of ninety-two putative MtGST genes that code for 120 proteins. All these members were classified into twelve classes based on their phylogenetic relationship and the presence of structural conserved domain/motif. Among them, 7 MtGST gene pairs were identified to have segmental duplication. Expression profiling of MtGST transcripts revealed their high level of organ/tissue-specific expression in most of the developmental stages and anatomical tissues. The transcripts of MtGSTU5, MtGSTU8, MtGSTU17, MtGSTU46, and MtGSTU47 showed significant up-regulation in response to various abiotic and biotic stresses. Moreover, transcripts of MtGSTU8, MtGSTU14, MtGSTU28, MtGSTU30, MtGSTU34, MtGSTU46 and MtGSTF8 were found to be highly upregulated in response to drought treatment for 24h and 48h. Among the highly stress-responsive MtGST members, MtGSTU17 showed strong affinity towards its conventional substrates reduced glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB) with the lowest binding energy of-5.7 kcal/mol and -6.5 kcal/mol, respectively. Furthermore, the substrate-binding site residues of MtGSTU17 were found to be highly conserved. These findings will facilitate the further functional and evolutionary characterization of GST genes in Medicago.

Diversity of Omega Glutathione Transferases in mushroom-forming fungi revealed by phylogenetic, transcriptomic, biochemical and structural approaches.

The Omega class of glutathione transferases (GSTs) forms a distinct class within the cytosolic GST superfamily because most of them possess a catalytic cysteine residue. The human GST Omega 1 isoform was first characterized twenty years ago, but it took years of work to clarify the roles of the human isoforms. Concerning the kingdom of fungi, little is known about the cellular functions of Omega glutathione transferases (GSTOs), although they are widely represented in some of these organisms. In this study, we re-assess the phylogeny and the classification of GSTOs based on 240 genomes of mushroom-forming fungi (Agaricomycetes). We observe that the number of GSTOs is not only extended in the order of Polyporales but also in other orders such as Boletales. Our analysis leads to a new classification in which the fungal GSTOs are divided into two Types A and B. The catalytic residue of Type-A is either cysteine or serine, while that of Type-B is cysteine. The present study focuses on Trametes versicolor GSTO isoforms that possess a catalytic cysteine residue. Transcriptomic data show that Type-A GSTOs are constitutive enzymes while Type-B are inducible ones. The crystallographic analysis reveals substantial structural differences between the two types while they have similar biochemical profiles in the tested conditions. Additionally, these enzymes have the ability to bind antioxidant molecules such as wood polyphenols in two possible binding sites as observed from X-ray structures. The multiplication of GSTOs could allow fungal organisms to adapt more easily to new environments.

A newly identified cluster of glutathione S-transferase genes provides Verticillium wilt resistance in cotton.

As the largest cultivated fiber crop in the world, cotton (Gossypium hirsutum) is often exposed to various biotic stresses during its growth periods. Verticillium wilt caused by Verticillium dahliae is a severe disease in cotton, and the molecular mechanism of cotton resistance for Verticillium wilt needs to be further investigated. Here, we revealed that the cotton genome contains nine types of GST genes. An evolutionary analysis showed that a newly identified cluster (including Gh_A09G1508, Gh_A09G1509 and Gh_A09G1510) located on chromosome 09 of the A-subgenome was under positive selection pressure during the formation of an allotetraploid. Transcriptome analysis showed that this cluster participates in Verticillium wilt resistance. Because the Gh_A09G1509 gene showed the greatest differential expression in the resistant cultivar under V. dahliae stress, we overexpressed this gene in tobacco and found that its overexpression resulted in enhanced Verticillium wilt resistance. Suppression of the gene cluster via virus-induced gene silencing made cotton plants of the resistant cultivar Nongda601 significantly susceptible. These results demonstrated that the GST cluster played an important role in Verticillium wilt resistance. Further investigation showed that the encoded enzymes of the cluster were essential for the delicate equilibrium between the production and scavenging of H2 O2 during V. dahliae stress.

Trametes versicolor glutathione transferase Xi 3, a dual Cys-GST with catalytic specificities of both Xi and Omega classes.

Glutathione transferases (GSTs) from the Xi and Omega classes have a catalytic cysteine residue, which gives them reductase activities. Until now, they have been assigned distinct substrates. While Xi GSTs specifically reduce glutathionyl-(hydro)quinones, Omega GSTs are specialized in the reduction of glutathionyl-acetophenones. Here, we present the biochemical and structural analysis of TvGSTX1 and TvGSTX3 isoforms from the wood-degrading fungus Trametes versicolor. TvGSTX1 reduces GS-menadione as expected, while TvGSTX3 reduces both Xi and Omega substrates. An in-depth structural analysis indicates a broader active site for TvGSTX3 due to specific differences in the nature of the residues situated in the C-terminal helix α9. This feature could explain the catalytic duality of TvGSTX3. Based on phylogenetic analysis, we propose that this duality might exist in saprophytic fungi and ascomycetes.

Crystal structure of the delta-class glutathione transferase in Musca domestica.

Among the various glutathione transferase (GST) isozymes in insects, the delta- and epsilon-class GSTs fulfill critical functions during the detoxification of insecticides. We crystalized MdGSTD1, the major delta-class GST isozyme in the housefly (Musca domestica), in complex with glutathione (GSH) and solved its structure at a resolution of 1.4 Å. The overall folding of MdGSTD1 resembled other known delta-class GSTs. Its substrate binding pocket was exposed to solvent and considerably more open than in the epsilon-class GST from M. domestica (MdGSTE2). However, their C-terminal structures differed the most because of the different lengths of the C-terminal regions. Although this region does not seem to directly interact with substrates, its deletion reduced the enzymatic activity by more than 70%, indicating a function in maintaining the proper conformation of the binding pocket. Binding of GSH to the GSH-binding region of MdGSTD1 results in a rigid conformation of this region. Although MdGSTD1 has a higher affinity for GSH than the epsilon class enzymes, the thiol group of the GSH molecule was not close enough to serine residue 9 to form a hydrogen-bond with this residue, which is predicted to act as the catalytic center for thiol group deprotonation in GSH.

Complex evolution of the GSTM gene family involves sharing of GSTM1 deletion polymorphism in humans and chimpanzees.

BACKGROUND: The common deletion of the glutathione S-transferase Mu 1 (GSTM1) gene in humans has been shown to be involved in xenobiotic metabolism and associated with bladder cancer. However, the evolution of this deletion has not been investigated. RESULTS: In this study, we conducted comparative analyses of primate genomes. We demonstrated that the GSTM gene family has evolved through multiple structural variations, involving gene duplications, losses, large inversions and gene conversions. We further showed experimentally that the GSTM1 was polymorphically deleted in both humans and also in chimpanzees, through independent deletion events. To generalize our results, we searched for genic deletions that are polymorphic in both humans and chimpanzees. Consequently, we found only two such deletions among the thousands that we have searched, one of them being the GSTM1 deletion and the other surprisingly being another metabolizing gene, the UGT2B17. CONCLUSIONS: Overall, our results support the emerging notion that metabolizing gene families, such as the GSTM, NAT, UGT and CYP, have been evolving rapidly through gene duplication and deletion events in primates, leading to complex structural variation within and among species with unknown evolutionary consequences.

Transcriptome profiling reveals differential gene expression of detoxification enzymes in a hemimetabolous tobacco pest after feeding on jasmonate-silenced Nicotiana attenuata plants.

BACKGROUND: The evolutionary arms race between plants and insects has driven the co-evolution of sophisticated defense mechanisms used by plants to deter herbivores and equally sophisticated strategies that enable phytophagous insects to rapidly detoxify the plant's defense metabolites. In this study, we identify the genetic determinants that enable the mirid, Tupiocoris notatus, to feed on its well-defended host plant, Nicotiana attenuata, an outstanding model for plant-insect interaction studies. RESULTS: We used an RNAseq approach to evaluate the global gene expression of T. notatus after feeding on a transgenic N. attenuata line which does not accumulate jasmonic acid (JA) after herbivory, and consequently accumulates very low levels of defense metabolites. Using Illumina sequencing, we generated a de novo assembled transcriptome which resulted in 63,062 contigs (putative transcript isoforms) contained in 42,610 isotigs (putative identified genes). Differential expression analysis based on RSEM-estimated transcript abundances identified 82 differentially expressed (DE) transcripts between T. notatus fed on wild-type and the defenseless plants. The same analysis conducted with Corset-estimated transcript abundances identified 59 DE clusters containing 85 transcripts. In both analyses, a larger number of DE transcripts were found down-regulated in mirids feeding on JA-silenced plants (around 70%). Among these down-regulated transcripts we identified seven transcripts possibly involved in the detoxification of N. attenuata defense metabolite, specifically, one glutathione-S-transferase (GST), one UDP-glucosyltransferase (UGT), five cytochrome P450 (P450s), and six serine proteases. Real-time quantitative PCR confirmed the down-regulation for six transcripts (encoding GST, UGT and four P450s) and revealed that their expression was only slightly decreased in mirids feeding on another N. attenuata transgenic line specifically silenced in the accumulation of diterpene glycosides, one of the many classes of JA-mediated defenses in N. attenuata. CONCLUSIONS: The results provide a transcriptional overview of the changes in a specialist hemimetabolous insect associated with feeding on host plants depleted in chemical defenses. Overall, the analysis reveals that T. notatus responses to host plant defenses are narrow and engages P450 detoxification pathways. It further identifies candidate genes which can be tested in future experiments to understand their role in shaping the T. notatus-N. attenuata interaction.

Clonorchis sinensis omega-class glutathione transferases play major roles in the protection of the reproductive system during maturation and the response to oxidative stress.

BACKGROUND: Clonorchis sinensis causes a major food-borne helminthic infection. This species locates in mammalian hepatobiliary ducts, where oxidative stressors and hydrophobic substances are profuse. To adapt to the hostile micromilieu and to ensure its long-term survival, the parasite continuously produces a diverse repertoire of antioxidant enzymes including several species of glutathione transferases (GSTs). Helminth GSTs play pertinent roles during sequestration of harmful xenobiotics since most helminths lack the cytochrome P-450 detoxifying enzyme. METHODS: We isolated and analyzed the biochemical properties of two omega-class GSTs of C. sinensis (CsGSTo1 and CsGSTo2). We observed spatiotemporal expression patterns in accordance with the maturation of the worm's reproductive system. Possible biological protective roles of CsGSTos in these organs under oxidative stress were investigated. RESULTS: The full-length cDNAs of CsGSTo1 and 2 constituted 965 bp and 1,061 bp with open reading frames of 737 bp (246 amino acids) and 669 bp (223 amino acids). They harbored characteristic N-terminal thioredoxin-like and C-terminal α-helical domains. A cysteine residue, which constituted omega-class specific active site, and the glutathione-binding amino acids, were recognized in appropriate positions. They shared 44 % sequence identity with each other and 14.8-44.8 % with orthologues/homologues from other organisms. Bacterially expressed recombinant proteins (rCsGSTo1 and 2) exhibited dehydroascorbate reductase (DHAR) and thioltransferase activities. DHAR activity was higher than thioltransferase activity. They showed weak canonical GST activity toward 1-chloro-2,4-dinitrobenzene. S-hexylglutathione potently and competitively inhibited the active-site at nanomolar concentrations (0.63 and 0.58 nM for rCsGSTo1 and 2). Interestingly, rCsGSTos exhibited high enzyme activity toward mu- and theta-class GST specific substrate, 4-nitrobenzyl chloride. Expression of CsGSTo transcripts and proteins increased beginning in 2-week-old juveniles and reached their highest levels in 4-week-old adults. The proteins were mainly expressed in the elements of the reproductive system, such as vitelline follicles, testes, seminal receptacle, sperm and eggs. Oxidative stressors induced upregulated expression of CsGSTos in these organs. Regardless of oxidative stresses, CsGSTos continued to be highly expressed in eggs. CsGSTo1 or 2 overexpressing bacteria demonstrated high resistance under oxidative killing. CONCLUSIONS: CsGSTos might be critically involved in protection of the reproductive system during maturation of C. sinensis worms and in response to oxidative conditions, thereby contributing to maintenance of parasite fecundity.

[Structure and functions of glutathione transferases].

Data about classification, nomenclature, structure, substrate specificity and role of many glutathione transferase's isoenzymes in cell functions have been summarised. The enzyme has been discovered more than 50 years ago. This family of proteins is updated continuously. It has very different composition and will have demand for system analysis for many years.

Glutathione transferase supergene family in tomato: Salt stress-regulated expression of representative genes from distinct GST classes in plants primed with salicylic acid.

A family tree of the multifunctional proteins, glutathione transferases (GSTs, EC 2.5.1.18) was created in Solanum lycopersicum based on homology to known Arabidopsis GSTs. The involvement of selected SlGSTs was studied in salt stress response of tomato primed with salicylic acid (SA) or in un-primed plants by real-time qPCR. Selected tau GSTs (SlGSTU23, SlGSTU26) were up-regulated in the leaves, while GSTs from lambda, theta, dehydroascorbate reductase and zeta classes (SlGSTL3, SlGSTT2, SlDHAR5, SlGSTZ2) in the root tissues under salt stress. Priming with SA exhibited a concentration dependency; SA mitigated the salt stress injury and caused characteristic changes in the expression pattern of SlGSTs only at 10(-4) M concentration. SlGSTF4 displayed a significant up-regulation in the leaves, while the abundance of SlGSTL3, SlGSTT2 and SlGSTZ2 transcripts were enhanced in the roots of plants primed with high SA concentration. Unexpectedly, under high salinity the SlDHAR2 expression decreased in primed roots as compared to the salt-stressed plants, however, the up-regulation of SlDHAR5 isoenzyme contributed to the maintenance of DHAR activity in roots primed with high SA. The members of lambda, theta and zeta class GSTs have a specific role in salt stress acclimation of tomato, while SlGSTU26 and SlGSTF4, the enzymes with high glutathione conjugating activity, characterize a successful priming in both roots and leaves. In contrast to low concentration, high SA concentration induced those GSTs in primed roots, which were up-regulated under salt stress. Our data indicate that induction of GSTs provide a flexible tool in maintaining redox homeostasis during unfavourable conditions.

Alpha-class glutathione S-transferases in wild turkeys (Meleagris gallopavo): characterization and role in resistance to the carcinogenic mycotoxin aflatoxin B1.

Domestic turkeys (Meleagris gallopavo) are one of the most susceptible animals known to the toxic effects of the mycotoxin aflatoxin B1 (AFB1), a potent human hepatocarcinogen, and universal maize contaminant. We have demonstrated that such susceptibility is associated with the inability of hepatic glutathione S-transferases (GSTs) to detoxify the reactive electrophilic metabolite exo-AFB1-8,9-epoxide (AFBO). Unlike their domestic counterparts, wild turkeys, which are relatively AFB1-resistant, possess hepatic GST-mediated AFBO conjugating activity. Here, we characterized the molecular and functional properties of hepatic alpha-class GSTs (GSTAs) from wild and domestic turkeys to shed light on the differences in resistance between these closely related strains. Six alpha-class GST genes (GSTA) amplified from wild turkeys (Eastern and Rio Grande subspecies), heritage breed turkeys (Royal Palm) and modern domestic (Nicholas strain) turkeys were sequenced, and catalytic activities of heterologously-expressed recombinant enzymes determined. Alpha-class identity was affirmed by conserved GST domains and four signature motifs. All GSTAs contained single nucleotide polymorphisms (SNPs) in their coding regions: GSTA1.1 (5 SNPs), GSTA1.2 (7), GSTA1.3 (3), GSTA2 (3), GSTA3 (1) and GSTA4 (2). E. coli-expressed GSTAs possessed varying activities toward GST substrates 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), ethacrynic acid (ECA), cumene hydroperoxide (CHP). As predicted by their relative resistance, livers from domestic turkeys lacked detectable GST-mediated AFBO detoxification activity, whereas those from wild and heritage birds possessed this critical activity, suggesting that intensive breeding and selection resulted in loss of AFB1-protective alleles during domestication. Our observation that recombinant tGSTAs detoxify AFBO, whereas their hepatic forms do not, implies that the hepatic forms of these enzymes are down-regulated, silenced, or otherwise modified by one or more mechanisms. These data may inform of possible molecular mechanisms of resistance to AFB1, and may also have the benefit of identifying genetic markers which could be used to enhance AFB1 resistance in modern domestic strains.

Sphingobium sp. SYK-6 LigG involved in lignin degradation is structurally and biochemically related to the glutathione transferase ω class.

SpLigG is one of the three glutathione transferases (GSTs) involved in the process of lignin breakdown in the soil bacterium Sphingobium sp. SYK-6. Sequence comparisons showed that SpLigG and several proteobacteria homologues form an independent cluster within cysteine-containing GSTs. The relationship between SpLigG and other GSTs was investigated. The X-ray structure and biochemical properties of SpLigG indicate that this enzyme belongs to the omega class of glutathione transferases. However, the hydrophilic substrate binding site of SpLigG, together with its known ability to stereoselectively deglutathionylate the physiological substrate α-glutathionyl-ß-hydroxypropiovanillone, argues for broadening the definition of the omega class.

Produccion de beta-glucosidasas por cultivos de bacterias termofilas indigenas del altiplano boliviano / beta-Glucoside production by thermophilic bacteria indigenous the bolivian altiplano culture

Las beta-glucosidasas son enzimas que poseen actividad hidrolitica y transferasa o transglucosidasa. Tienen diversas aplicaciones; en la biosintesis de oligosacaridos, produccion de etanol utilizando residuos agricolas y en la industria de vinos. La aplicacion industrial, sin embargo, requiere estabilidad a temperaturas elevadas, por lo que los microorganismos termofilos tienen gran interes. El proposito de esta investigacion es el de optimizar el medio de cultivo anaerobio de bacterias termofilas, para aumentar la produccion de beta-glucosidasas. Esta enzima es producida por tres aislados bacterianos: FT3, 2B y P5 los cuales fueron aislados de la region andina de Bolivia. El aislado bacteriano FT3 mostro una actividad beta-glucosidasa de 0,35 [UI/mL]. Se tomaron como variables dentro de la optimizacion del medio de cultivo las fuentes de nitrogeno y de carbono, y el pH. Asi tambien se probaron dos sistemas de cultivo: celulas libres y encapsuladas. Empleando extracto de levadura como fuente de nitrogeno se obtuvo una actividad de 0,52 [UI/mL]. En la optimizacion del pH del medio de cultivo se obtuvo una actividad de 0,81 [UI/mL] a pH 5. Como fuente de carbono se eligieron los hidrolizados de paja de trigo y paja de quinoa lleg¨¢ndose a obtener actividades de 1,27 y 1,34 [UI/mL] respectivamente. Se establecio que la localizacion celular de la enzima beta-glucosidasa es extracelular y presenta estabilidad hasta una temperatura de 80 ºC y un pH de 7.

The beta-glucosidases possess hydrolytic and transferase activity or transglucosidase. They have various applications; such as biosynthesis of oligosaccharides, production of ethanol using agricultural residues and wine industry. However for industrial application, stability to high temperatures is needed. Therefore a great interesting in the thermophile microorganism study exist. The purpose of this research is to optimize the culture medium of thermophilic anaerobic bacteria to increase the production of beta-glucosidase. This enzyme is produced by three isolate bacterial FT3, 2B and P5 which were isolated from the Andean region of Bolivia. FT3 isolate showed beta-glucosidase activity of 0.35 [IU/mL]. In regards to the optimization of culture medium variables such as nitrogen source, carbon source and pH were taken into account and also the combination with free and encapsulated bacterial cells. Yeast extract was the selected source of nitrogen obtaining an activity of 0.52 [IU/ mL]. The optimal pH was 5 obtaining an activity of 0.81 [IU/mL]. The selected carbon source was the hydrolyzed wheat straw and quinoa straw obtaining activities of 1.27 and 1.34 [IU/mL], respectively. The cellular localization of beta-glucosidase enzyme is extracellular and provides stability to temperature of 80 ºC and stability at pH 7.

Response of glutathione S-transferase (GST) genes to cadmium exposure in the marine pollution indicator worm, Perinereis nuntia.

Glutathione S-transferase (GST) is a phase II enzyme that functions as a detoxicant by catalyzing the conjugation of reduced glutathione with a variety of xenobiotics via cysteine thiol. Molecular genetic approaches using gene biomarkers show substantial relevance as sensitive biomarkers for the indication of pollution levels. In order to use GSTs as molecular biomarkers for marine pollution monitoring, we cloned and sequenced the full-length cDNA of seven GST genes from the marine polychaete Perinereis nuntia. The deduced amino acid sequence of Pn-GSTs showed a high similarity to those of other species that clustered into the same clades in a phylogenetic analysis. In addition, to evaluate Pn-GSTs as useful biomarkers on effects after cadmium (Cd) exposure, we exposed sublethal concentrations of Cd (5, 50, and 500 µg/L) to P. nuntia, and they showed relatively different but significantly increases, depending on exposure time and Cd concentrations. Particularly, Pn-GST-omega and Pn-GST-sigma genes were highly sensitive with a clear dose-dependent manner on mRNA expression. The total GST activities also have significantly increased levels at higher concentrations of Cd exposure. These results indicate that Pn-GSTs play important roles in Cd-induced oxidative stress in terms of the physiological changes relating to metabolism and cell protection, and those genes would have great potential as molecular biomarkers to monitor marine environmental health.

Glutathione transferases of Phanerochaete chrysosporium: S-glutathionyl-p-hydroquinone reductase belongs to a new structural class.

The white rot fungus Phanerochaete chrysosporium, a saprophytic basidiomycete, possesses a large number of cytosolic glutathione transferases, eight of them showing similarity to the Omega class. PcGSTO1 (subclass I, the bacterial homologs of which were recently proposed, based on their enzymatic function, to constitute a new class of glutathione transferase named S-glutathionyl-(chloro)hydroquinone reductases) and PcGSTO3 (subclass II related to mammalian homologs) have been investigated in this study. Biochemical investigations demonstrate that both enzymes are able to catalyze deglutathionylation reactions thanks to the presence of a catalytic cysteinyl residue. This reaction leads to the formation of a disulfide bridge between the conserved cysteine and the removed glutathione from their substrate. The substrate specificity of each isoform differs. In particular PcGSTO1, in contrast to PcGSTO3, was found to catalyze deglutathionylation of S-glutathionyl-p-hydroquinone substrates. The three-dimensional structure of PcGSTO1 presented here confirms the hypothesis that it belongs not only to a new biological class but also to a new structural class that we propose to name GST xi. Indeed, it shows specific features, the most striking ones being a new dimerization mode and a catalytic site that is buried due to the presence of long loops and that contains the catalytic cysteine.

Investigation of the role of conserved residues Ser13, Asn48 and Pro49 in the catalytic mechanism of the tau class glutathione transferase from Glycine max.

Plant glutathione transferases (GSTs) play a key role in the metabolism of various xenobiotics. In this report, the catalytic mechanism of the tau class GSTU4-4 isoenzyme from Glycine max (GmGSTU4-4) was investigated by site-directed mutagenesis and steady-state kinetic analysis. The catalytic properties of the wild-type enzyme and three mutants of strictly conserved residues (Ser13Ala, Asn48Ala and Pro49Ala) were studied in 1-chloro-2,4-dinitrobenzene (CDNB) conjugation reaction. The results showed that the mutations significantly affect substrate binding and specificity. The effect of Ser13Ala mutation on the catalytic efficiency of the enzyme could be explained by assuming the direct involvement of Ser13 to the reaction chemistry and the correct positioning of GSH and CDNB in the ternary catalytic complex. Asn48 and Pro49 were found to have a direct role on the structural integrity of the GSH-binding site (G-site). Moreover, mutation of Asn48 and Pro49 residues may bring about secondary effects altering the thermal stability and the catalytic activity (k(cat)) of the enzyme without affecting the nature of the rate-limiting step of the catalytic reaction.

Glutathione transferases are structural and functional outliers in the thioredoxin fold.

Glutathione transferases (GSTs) are ubiquitous scavengers of toxic compounds that fall, structurally and functionally, within the thioredoxin fold suprafamily. The fundamental catalytic capability of GSTs is catalysis of the nucleophilic addition or substitution of glutathione at electrophilic centers in a wide range of small electrophilic compounds. While specific GSTs have been studied in detail, little else is known about the structural and functional relationships between different groupings of GSTs. Through a global analysis of sequence and structural similarity, it was determined that variation in the binding of glutathione between the two major subgroups of cytosolic (soluble) GSTs results in a different mode of glutathione activation. Additionally, the convergent features of glutathione binding between cytosolic GSTs and mitochondrial GST kappa are described. The identification of these structural and functional themes helps to illuminate some of the fundamental contributions of the thioredoxin fold to catalysis in the GSTs and clarify how the thioredoxin fold can be modified to enable new functions.

Glutathione S-transferase expression and isoenzyme composition during cell differentiation of Caco-2 cells.

The human colon adenocarcinoma cell line Caco-2 is frequently used to study human intestinal metabolism and transport of xenobiotica. Previous studies have shown that both Caco-2 cells and human colon cells constitutively express the multigene family of detoxifying enzymes glutathione S-transferases (GSTs), particularly GST alpha and GST pi. GSTs may play a fundamental role in the molecular interplay between phase I, II enzymes and ABC-transporters. The gut fermentation product, butyrate, can modulate the potential for detoxification. The aim of this study was to investigate the basal expression of further cytosolic GSTs in Caco-2 cells during cell differentiation. In addition, a comparison was made with expression levels in MCF-7 and HepG2, two other cell types with barrier functions. Finally, the butyrate-mediated modulation of gene and protein expression was determined by real time PCR and western blot analysis. In Caco-2, gene and protein expression levels of GST alpha increased during cell differentiation. High levels of GSTO1 and GSTP1 were constantly expressed. No expression of GSTM5 and GSTT1 was detected. HepG2 expressed GSTO1 and MCF-7 GSTZ1 most intensively. No expression of GSTA5, GSTM5, or GSTP1 was detected in either cell. Incubation of Caco-2 cells with butyrate (5 mM) significantly induced GSTA1 and GSTM2 in proliferating Caco-2 cells. In differentiated cells, butyrate tended to increase GSTO1 and GSTP1. The results of this study show that a differentiation-dependent expression of GSTs in Caco-2 cells may reflect the in vivo situation and indicate the potential of butyrate to modify intestinal metabolism. GSTA1-A4 have been identified as good markers for cell differentiation. The Caco-2 cell line is a useful model for assessing the potential of food-related substances to modulate the GST expression pattern.

Novel omega glutathione S-transferases in disk abalone: Characterization and protective roles against environmental stress.

Omega glutathione S-transferases (GSTs) are a newly identified class of GSTs with unique properties compared to other members in GST superfamily. This present study reports the cloning, characterization and stress-induced expression analysis of two omega GST genes in disk abalone, Haliotis discus discus. Two disk abalone omega GST genes, HdGSTO1 and HdGSTO2, encode two polypeptides with calculated molecular mass of 27.4 and 26.9 kDa, respectively. Their deduced amino acid sequences showed highest similarity with another molluscan omega GST from Crassostrea gigas. Three-dimensional structures of two omega GSTs were generated by homology modeling and exhibited typical omega GST structural characteristics. The recombinant proteins of HdGSTO1 and HdGSTO2 showed glutathione-dependent thioltransferase and dehydroascorbate reductase activities; however, no activity towards other common GST substrates was detected. Of the two genes, protein encoded by HdGSTO1 showed much higher catalytic ability than the other one. HdGSTO1 mRNA was expressed ubiquitously with high levels in all examined tissues, while HdGSTO2 showed specific expression in gonad and digestive tract. The transcriptional levels of HdGSTO1 in gill were dramatically elevated when abalones were subjected to heat shock, heavy metals and endocrine-disrupting chemical (EDC) exposure, indicating that HdGSTO1 might play important protective roles against environmental stress. HdGSTO2 expression was also significantly induced by heavy metals and EDCs although with much lower fold change than HdGSTO1. But under thermal stress, HdGSTO2 expression was repressed in a time-dependent pattern, implying its different physiological roles under stress. These results indicate that omega GSTs of the disk abalone, especially HdGSTO1, have great potentials as highly sensitive biomarkers of environmental stress.

Crystal structure of Glycine max glutathione transferase in complex with glutathione: investigation of the mechanism operating by the Tau class glutathione transferases.

Cytosolic GSTs (glutathione transferases) are a multifunctional group of enzymes widely distributed in Nature and involved in cellular detoxification processes. The three-dimensional structure of GmGSTU4-4 (Glycine max GST Tau 4-4) complexed with GSH was determined by the molecular replacement method at 2.7 A (1 A=0.1 nm) resolution. The bound GSH is located in a region formed by the beginning of alpha-helices H1, H2 and H3 in the N-terminal domain of the enzyme. Significant differences in the G-site (GSH-binding site) as compared with the structure determined in complex with Nb-GSH [S-(p-nitrobenzyl)-glutathione] were found. These differences were identified in the hydrogen-bonding and electrostatic interaction pattern and, consequently, GSH was found bound in two different conformations. In one subunit, the enzyme forms a complex with the ionized form of GSH, whereas in the other subunit it can form a complex with the non-ionized form. However, only the ionized form of GSH may form a productive and catalytically competent complex. Furthermore, a comparison of the GSH-bound structure with the Nb-GSH-bound structure shows a significant movement of the upper part of alpha-helix H4 and the C-terminal. This indicates an intrasubunit modulation between the G-site and the H-site (electrophile-binding site), suggesting that the enzyme recognizes the xenobiotic substrates by an induced-fit mechanism. The reorganization of Arg111 and Tyr107 upon xenobiotic substrate binding appears to govern the intrasubunit structural communication between the G- and H-site and the binding of GSH. The structural observations were further verified by steady-state kinetic analysis and site-directed mutagenesis studies.