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.     

Selective Binding of Glutathione Conjugates of Fatty Acid Derivatives by Plant Glutathione Transferases

Proteomic studies with Arabidopsis thaliana have revealed that the plant-specific Tau (U) class glutathione transferases (GSTs) are selectively retained by S-hexylglutathione affinity supports. Overexpression of members of the Arabidopsis GST superfamily in Escherichia coli showed that 25 of the complement of 28 GSTUs caused the aberrant accumulation of acylated glutathione thioesters in vivo, a perturbation that was not observed with other GST classes. Each GSTU caused a specific group of fatty acyl derivatives to accumulate, which varied in chain length (C₆ to C₁₈), additional oxygen content (0 or 1), and desaturation (0 or 1). Thioesters bound tightly to recombinant GSTs (K_d ∼ 1 μm), explaining their accumulation. Transient expression of GSTUs in Nicotiana benthamiana followed by recovery by Strep-tag affinity chromatography allowed the respective plant ligands to be extracted and characterized. Again, each GST showed a distinct profile of recovered metabolites, notably glutathionylated oxophytodienoic acid and related oxygenated fatty acids. Similarly, the expression of the major Tau protein GSTU19 in the endogenous host Arabidopsis led to the selective binding of the glutathionylated oxophytodienoic acid-glutathione conjugate, with the enzyme able to catalyze the conjugation reaction. Additional ligands identified in planta included other fatty acid derivatives including divinyl ethers and glutathionylated chlorogenic acid. The strong and specific retention of various oxygenated fatty acids by each GSTU and the conservation in binding observed in the different hosts suggest that these proteins have selective roles in binding and conjugating these unstable metabolites in vivo.In plants, the glutathione transferases (GSTs²; EC 2.5.1.18) are a superfamily of proteins with the dominant Phi (F) and Tau (U) classes having largely undefined functions in endogenous metabolism (1). This is in contrast to their relatively well studied role in catalyzing the glutathione conjugation of herbicides (2). Because of their importance in determining herbicide selectivity, these proteins have been purified from a range of major crops including maize (3), soybean (4), and wheat (5, 6). One approach which has been commonly applied to isolate these proteins is affinity chromatography using a range of glutathione (GSH) derivatives as ligands (7). These ligands often display a surprising degree of selectivity in capturing specific GSTs. Thus, in wheat extracts, whereas GSH-agarose effectively isolated GSTFs, S-hexylglutathione (GS-hexyl) proved to be a selective ligand for GSTUs (5). Similarly, glutathionylated dyes have proved to be highly selective for specific GSTs in maize (8). In each application, the use of GSH derivatives as affinity ligands has been developed through serendipity, with limited attention directed to the potential significance of such binding selectivity in relating to the nature of in vivo substrates and reaction products. At a molecular level, crystallographic studies with both plant GSTFs (9) and GSTUs (10) have revealed a conserved active site architecture consisting of a GSH binding domain that orientates the thiol toward an adjacent hydrophobic binding pocket containing the putative alkyl- or aryl-bearing acceptor. However, the nature of any such hydrophobic substrates in plants remains largely unknown, with little evidence that natural products accumulate as glutathione conjugates (11).To address the significance of selective binding to glutathionylated derivatives, members of the GST superfamily from Arabidopsis thaliana have been screened for their ability to bind conjugated metabolites in vivo. Two methods have been used. In the first approach, the GSTs in Arabidopsis plants which bind GS-hexyl have been identified using affinity chromatography coupled with proteomics. In the second approach, the ligands which associate with these enzymes in vivo have been identified in a two-tiered screen. In the first instance, GSTs have been individually expressed in Escherichia coli, and the crude extracts from the recombinant bacteria were assessed for their aberrant accumulation of alkylated glutathione derivatives. Here, we reasoned that the lack of compartmentalization in bacteria would allow the GST to interact with any endogenous metabolic intermediate with disruptions in metabolism then directly monitored. A similar approach has recently demonstrated that specific maize GSTUs derail heme metabolism in recombinant E. coli by selectively binding porphyrinogen intermediates (12). In the secondary screening, we have then taken Strep-tagged GSTs with interesting profiles of ligand binding activity in E. coli and transgenically overexpressed them in Nicotiana benthamiana and/or Arabidopsis. The proteins were then recovered using the affinity tag, and bound ligands were identified by HPLC-MS.     

Comparative genomics identifies new alpha class genes within the avian glutathione S-transferase gene cluster

Glutathione S-transferases (GSTs: EC2.5.1.18) are a superfamily of multifunctional dimeric enzymes that catalyze the conjugation of glutathione (GSH) to electrophilic chemicals. In most animals and in humans, GSTs are the principal enzymes responsible for detoxifying the mycotoxin aflatoxin B₁ (AFB₁) and GST dysfunction is a known risk factor for susceptibility towards AFB₁. Turkeys are one of the most susceptible animals known to AFB₁, which is a common contaminant of poultry feeds. The extreme susceptibility of turkeys is associated with hepatic GSTs unable to detoxify the highly reactive and electrophilic metabolite exo-AFB₁-8,9-epoxide (AFBO). In this study, comparative genomic approaches were used to amplify and identify the α-class tGST genes (tGSTA1.1, tGSTA1.2, tGSTA1.3, tGSTA2, tGSTA3 and tGSTA4) from turkey liver. The conserved GST domains and four α-class signature motifs in turkey GSTs (with the exception of tGSTA1.1 which lacked one motif) confirm the presence of hepatic α-class GSTs in the turkey. Four signature motifs and conserved residues found in α-class tGSTs are (1) xMExxxWLLAAAGVE, (2) YGKDxKERAxIDMYVxG, (3) PVxEKVLKxHGxxxL and (4) PxIKKFLXPGSxxKPxxx. A BAC clone containing the α-class GST gene cluster was isolated and sequenced. The turkey α-class GTS genes genetically map to chromosome MGA2 with synteny between turkey and human α-class GSTs and flanking genes. This study identifies the α-class tGST gene cluster and genetic markers (SNPs, single nucleotide polymorphisms) that can be used to further examine AFB₁ susceptibility and resistance in turkeys. Functional characterization of heterologously expressed proteins from these genes is currently underway.     

Conjugation of the linoleic acid oxidation product, 13-oxooctadeca-9,11-dienoic acid,a bioactive endogenous substrate for mammalian glutathione transferase

The oxidation of linoleic acid leads to the generation of several products with biological activity, including 13-oxooctadeca-9,11-dienoic acid (13-OXO), a bioactive 2,4-dienone that has been linked to cell differentiation. In the current work, the conjugation of 13-OXO by human glutathione transferases (GSTs) of the alpha (A1–1, A4–4), mu (M1–1, M2–2) and pi (the allelic variants P1–1/ile, and P1–1/val) classes, and a rat theta (rT2–2) class enzyme has been evaluated. The kinetics and stereoselectivity of the production of the 13-OXO-glutathione conjugate (13-OXO-SG) have been examined. In contrast to many xenobiotic substrates, the endogenous substrate 13-OXO does not exhibit an appreciable non-enzymatic rate of conjugation under physiological conditions. Therefore, the GST-catalyzed conjugation takes on greater significance as it provides the only realistic means for formation of 13-OXO-SG in most biological systems. Alpha class enzymes are most efficient at catalyzing the formation of 13-OXO-SG with k_cat/K_m values of 8.9 mM⁻¹ s⁻¹ for GST A1–1 and 2.14 mM⁻¹ s⁻¹ for GST A4–4. In comparison, enzymes from the mu and pi classes exhibit specificity constants from 0.4 to 0.8 mM⁻¹ s⁻¹. Conjugation of 13-OXO with glutathione at C-9 of the substrate can yield a pair of diastereomers that can be resolved by chiral HPLC. GSTs from the mu and pi classes are the most stereoselective enzymes and there is no apparent relationship between catalytic efficiency and stereoselectivity. The role of GST in the metabolic disposition of the bioactive oxidation products of linoleic acid has implications for the regulation of normal cellular functions by these versatile enzymes.     

Inhibitory effect of metals on animal and plant glutathione transferases

Glutathione transferases (GSTs) represent a widespread enzyme superfamily in eukaryotes and prokaryotes catalyzing different reactions with endogenous and xenobiotic substrates such as organic pollutants. The latter are often found together with metal contamination in the environment. Besides performing of essential functions, GSTs protect cells by conjugation of glutathione with various reactive electrophiles. The interference of toxic metals with this functionality of GSTs may have unpredictable toxicological consequences for the organisms. In this review results from the recent literature are summarized and discussed describing the ability of metals to inhibit intracellular detoxification processes in animals and plants.     

Roles for Stress-inducible Lambda Glutathione Transferases in Flavonoid Metabolism in Plants as Identified by Ligand Fishing

The glutathione transferases (GSTs) of plants are a superfamily of abundant enzymes whose roles in endogenous metabolism are largely unknown. For example, the lambda class of GSTs (GSTLs) have members that are selectively induced by chemical stress treatments and based on their enzyme chemistry are predicted to have roles in redox homeostasis. However, using conventional approaches these functions have yet to be determined. To address this, recombinant GSTLs from wheat and Arabidopsis were tagged with a Strep tag and after affinity-immobilization, incubated with extracts from Arabidopsis, tobacco, and wheat. Bound ligands were then recovered by solvent extraction and identified by mass spectrometry (MS). With the wheat enzyme TaGSTL1, the ligand profiles obtained with in vitro extracts from tobacco closely matched those observed after the protein had been expressed in planta, demonstrating that these associations were physiologically representative. The stress-inducible TaGSTL1 was found to selectively recognize flavonols (e.g. taxifolin; K_d = 25 nm), with this binding being dependent upon S-glutathionylation of an active site cysteine. In the case of the wheat extracts, this selectivity in ligand recognitions lead to the detection of flavonols that had not been previously described in this cereal. Subsequent in vitro assays showed that the co-binding of flavonols, such as quercetin, to the thiolated TaGSTL1 represented an intermediate step in the reduction of the respective S-glutathionylated quinone derivatives to yield free flavonols. These results suggest a novel role for GSTLs in maintaining the flavonoid pool under stress conditions.     

The Biosynthesis of Thiol- and Thioether-containing Cofactors and Secondary Metabolites Catalyzed by Radical S-Adenosylmethionine Enzymes

Sulfur atoms are present as thiol and thioether functional groups in amino acids, coenzymes, cofactors, and various products of secondary metabolic pathways. The biosynthetic pathways for several sulfur-containing biomolecules require the substitution of sulfur for hydrogen at unreactive aliphatic or electron-rich aromatic carbon atoms. Examples discussed in this review include biotin, lipoic acid, methylthioether modifications found in some nucleic acids and proteins, and thioether cross-links found in peptide natural products. Radical S-adenosyl-l-methionine (SAM) enzymes use an iron-sulfur cluster to catalyze the reduction of SAM to methionine and a highly reactive 5′-deoxyadenosyl radical; this radical can abstract hydrogen atoms at unreactive positions, facilitating the introduction of a variety of functional groups. Radical SAM enzymes that catalyze sulfur insertion reactions contain a second iron-sulfur cluster that facilitates the chemistry, either by donating the cluster''s endogenous sulfide or by binding and activating exogenous sulfide or sulfur-containing substrates. The use of radical chemistry involving iron-sulfur clusters is an efficient anaerobic route to the generation of carbon-sulfur bonds in cofactors, secondary metabolites, and other natural products.     

6种植物次生物质对斜纹夜蛾解毒酶活性的影响

草食性昆虫取食植物时遇到宿主植物中大量次生物质的化学防御,研究昆虫适应植物毒素的反防御策略具有重要的科学意义。分别添加0.01%肉桂酸、0.01%水杨酸、0.01%花椒毒素、0.02%槲皮素、0.05%黄酮和0.1%香豆素等6种植物次生物质的人工饲料饲养斜纹夜蛾(Spodoptera litura)五龄幼虫48 h后,测定斜纹夜蛾幼虫中肠和脂肪体中谷胱甘肽S-转移酶(GSTs)、羧酸酯酶(CarE)、P450的酶含量及头部乙酰胆碱酯酶(AChE)的活性,利用半定量RT-PCR检测中肠和脂肪体中CYP4M14和CYP4S9的基因表达水平。结果表明:取食肉桂酸和香豆素后,斜纹夜蛾中肠中CarE的酶活性分别提高了1.67和1.37倍,取食6种次生物质均能显著提高斜纹夜蛾脂肪体中GSTs酶活性。取食肉桂酸和香豆素48 h后,脂肪体中P450酶含量比对照增加2.93和14.50倍。取食肉桂酸、花椒毒素、槲皮素和香豆素后,斜纹夜蛾头部AchE酶活性与对照相比提高了1.53、1.80、2.36和1.56倍。6种次生物质均可诱导脂肪体中CYP4M14基因表达,槲皮素、肉桂酸和香豆素强烈诱导CYP4S9在脂肪体中表达。表明,斜纹夜蛾具有利用植物次生物质诱导其解毒酶的能力,进而提高其对毒素的抗性。     

The role of GSTs in the tolerance of Rhizobium leguminosarum to cadmium

A high intraspecific difference in cadmium (Cd) tolerance exits among Rhizobium leguminosarum strains. The higher tolerance to Cd appeared to be related to the efficiency of the glutathione (GSH)–Cd chelation mechanism, but it is not known how efficiency is influenced. Thus, in this work it was intended to investigate the traits behind the efficiency of intracellular Cd chelation by GSH. Glutathione-S-transferases (GST; EC 2.5.1.18) are a family of multi-functional dimeric proteins, found in both prokaryotes and eukaryotes, which are implicated in a variety of stress conditions. The common feature of these enzymes is to catalyze the conjugation of the sulfur atom of GSH with a large variety of hydrophobic toxic compounds of both endogenous and exogenous origin. Taking into account the reactions catalyzed by GSTs, it was hypothesized that they could be involved in the GSH–Cd complex formation in R. leguminosarum. Differences in GSTs activity between strains could explain variation in Cd chelation efficiency detected among strains and, consequently, discrepancy in tolerance to Cd. Thus, GST isoforms of R. leguminosarum strains with distinct tolerances to Cd were purified and their activity investigated. The relationship between chelation efficiency and enzymatic activity of GSTs was demonstrated, supporting the hypothesis that GSTs, in particular one isoform, was involved in the formation of GSH–Cd complexes and in the tolerance of Rhizobium to Cd.     

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.     

An enzyme catalysing the conjugation of epoxides with glutathione

1. Liver supernatant preparations from rats and ferrets catalyse the conjugation of some epoxides with glutathione. The enzyme involved might be called `glutathione S-epoxidetransferase', as it is different from glutathione S-aryltransferase, the enzyme catalysing the conjugation of 1,2-dichloro-4-nitrobenzene, 4-nitro-pyridine N-oxide and other cyclic compounds with glutathione and from the enzyme catalysing the conjugation of iodomethane and glutathione. 2. The enzyme does not catalyse the reaction with cysteine. It is not inactivated by dialysis but is unstable at pH 5·0. 3. The role of the enzyme in metabolism of foreign compounds is discussed.     

Conjugation of the linoleic acid oxidation product, 13-oxooctadeca-9,11-dienoic acid,a bioactive endogenous substrate for mammalian glutathione transferase

The oxidation of linoleic acid leads to the generation of several products with biological activity, including 13-oxooctadeca-9,11-dienoic acid (13-OXO), a bioactive 2,4-dienone that has been linked to cell differentiation. In the current work, the conjugation of 13-OXO by human glutathione transferases (GSTs) of the alpha (A1-1, A4-4), mu (M1-1, M2-2) and pi (the allelic variants P1-1/ile, and P1-1/val) classes, and a rat theta (rT2-2) class enzyme has been evaluated. The kinetics and stereoselectivity of the production of the 13-OXO-glutathione conjugate (13-OXO-SG) have been examined. In contrast to many xenobiotic substrates, the endogenous substrate 13-OXO does not exhibit an appreciable non-enzymatic rate of conjugation under physiological conditions. Therefore, the GST-catalyzed conjugation takes on greater significance as it provides the only realistic means for formation of 13-OXO-SG in most biological systems. Alpha class enzymes are most efficient at catalyzing the formation of 13-OXO-SG with kcat/Km values of 8.9 mM(-1) s(-1) for GST A1-1 and 2.14 mM(-1) s(-1) for GST A4-4. In comparison, enzymes from the mu and pi classes exhibit specificity constants from 0.4 to 0.8 mM(-1) s(-1). Conjugation of 13-OXO with glutathione at C-9 of the substrate can yield a pair of diastereomers that can be resolved by chiral HPLC. GSTs from the mu and pi classes are the most stereoselective enzymes and there is no apparent relationship between catalytic efficiency and stereoselectivity. The role of GST in the metabolic disposition of the bioactive oxidation products of linoleic acid has implications for the regulation of normal cellular functions by these versatile enzymes.     

A Novel Quasi-Species of Glutathione Transferase with High Activity towards Naturally Occurring Isothiocyanates Evolves from Promiscuous Low-Activity Variants

Glutathione transferases (GSTs) are known as promiscuous enzymes capable of catalyzing the conjugation of glutathione with a broad range of electrophilic substrates. A previous study based on recombinant chimeras derived from human GST M1-1 and GST M2-2 demonstrated the formation of a subset of F1 generation GSTs, which had lost high activity with substrates distinguishing parental enzymes. In the present study, the members of this subset were recombined by DNA shuffling to produce an F2 generation of GSTs. Screening of 930 bacterial clones demonstrated that 83% of recombinant enzyme variants were active with at least one of three alternative substrates: phenethyl isothiocyanate (PEITC), 1-chloro-2,4-dinitrobenzene, or p-nitrophenyl acetate. The majority had similar low activity as the parental GSTs in the F1 generation. However, 17 novel enzymes displayed high activity with PEITC. Half of these enzymes were similar to GST M1-1, which also has high activity with the same substrate, and all of these GSTs featured Tyr116/Ser210 in the active site. This group of F2 variants apparently had reverted to the GST M1-1 type. A second group of F2 variants with high PEITC activity was characterized by His116 in the active site. This category represented a new variety of GSTs, which demonstrated higher selectivity for isothiocyanate substrates than the GST M1-1 type. The different groups of GSTs can be considered as distinct molecular quasi-species, each of which comprises variant amino acid sequences. The quasi-species are structurally distinguished by active-site residues that govern their substrate selectivities. Clearly, minimal alterations of the active site can generate enzymes with highly distinctive functional properties.     

Formation and biliary excretion of glutathione conjugates of bile acids in the rat as shown by liquid chromatography/electrospray ionization-linear ion trap mass spectrometry

Acyl-adenylates and acyl-CoA thioesters of bile acids (BAs) are reactive acyl-linked metabolites that have been shown to undergo transacylation-type reactions with the thiol group of glutathione (GSH), leading to the formation of thioester-linked GSH conjugates. In the current study, we examined the transformation of cholyl-adenylate (CA-AMP) and cholyl-coenzyme A thioester (CA-CoA) into a cholyl-S-acyl GSH (CA-GSH) conjugate by rat hepatic glutathione S-transferase (GST). The reaction product was analyzed by liquid chromatography (LC)/electrospray ionization (ESI)-linear ion trap mass spectrometry (MS). The GST-catalyzed formation of CA-GSH occurred with both CA-AMP and CA-CoA. Ursodeoxycholic acid, lithocholic acid, and 2,2,4,4-²H₄-labeled lithocholic acid were administered orally to biliary fistula rats, and their corresponding GSH conjugates were identified in bile by LC/ESI-MS². These in vitro and in vivo studies confirm a new mode of BA conjugation in which BAs are transformed into their GSH conjugates via their acyl-linked intermediary metabolites by the catalytic action of GST in the liver, and the GSH conjugates are then excreted into the bile.     

Glutathione S-transferases from the gastrointestinal nematode Heligmosomoides polygyrus and mammalian liver compared

Glutathione S-transferases have been partially characterised from the gastrointestinal nematode Heligmosomoides polygyrus. Two major subunit families were purified (24 and 23 kDa) with N-terminal homology to the mammalian Alpha family. Four dimeric forms of GST were purified from the nematode by glutathione-affinity chromatography, two major enzymes (pI 8.1, 5.0) and two minor forms (pI 5.8, 5.3). The purified GST pool could neutralize model and lipid peroxides via peroxidase activity but not peroxidation derived reactive carbonyls via glutathione transferase activity. Antisera raised to the pooled nematode GSTs appeared to recognize other Strongylida GSTs more strongly on Western blotting compared to mammalian GSTs.     

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.     

Diversification of Fungal Specific Class A Glutathione Transferases in Saprotrophic Fungi

Glutathione transferases (GSTs) form a superfamily of multifunctional proteins with essential roles in cellular detoxification processes and endogenous metabolism. The distribution of fungal-specific class A GSTs was investigated in saprotrophic fungi revealing a recent diversification within this class. Biochemical characterization of eight GSTFuA isoforms from Phanerochaete chrysosporium and Coprinus cinereus demonstrated functional diversity in saprotrophic fungi. The three-dimensional structures of three P. chrysosporium isoforms feature structural differences explaining the functional diversity of these enzymes. Competition experiments between fluorescent probes, and various molecules, showed that these GSTs function as ligandins with various small aromatic compounds, derived from lignin degradation or not, at a L-site overlapping the glutathione binding pocket. By combining genomic data with structural and biochemical determinations, we propose that this class of GST has evolved in response to environmental constraints induced by wood chemistry.     

Effect of copper exposure on GST activity and on the expression of four GSTs under oxidative stress condition in the monogonont rotifer, Brachionus koreanus

Glutathione S-transferases (GSTs; EC 2.5.1.18) are major enzymes that function in Phase II detoxification reactions by catalyzing the conjugation of reduced glutathione through cysteine thiol. In this study, we cloned and sequenced four GST genes from the monogonont rotifer Brachionus koreanus. The domain regions of four Bk-GSTs showed a high similarity to those of other species. In addition, to evaluate the potential of GST genes as an early warning signal for oxidative stress, we exposed sublethal concentrations of copper (Cu) to B. koreanus and measured glutathione (GSH) contents and several antioxidant enzymes such as glutathione S-transferase (GST), glutathione peroxidase (GPx; EC 1.11.1.9), and glutathione reductase (GR; EC 1.8.1.7). The reactive oxygen species (ROS) at 12 h and 24 h after copper exposure increased significantly. GSH contents however did not increase significantly and even it decreased at 0.24 mg/L at 12 h. The activities of several antioxidant enzymes, particularly GPx and GR, showed a dramatic increase in 0.24 mg/L of CuCl₂. Messenger RNAs of each Bk-GST showed different patterns of modulations according to GST types, and particularly, Bk-GST-omega, Bk-GST-sigma, and Bk-GST zeta genes were highly sensitive to Cu. These results indicate that Bk-GSTs, functioning as one of the enzymatic defense mechanisms particularly in the early stage of oxidative stress response, were induced by Cu exposure. This also suggests that these genes and related enzymes have a potential as biomarkers for a more sensitive initial stress response.     

Schisandra fructus extract ameliorates doxorubicin-induce cytotoxicity in cardiomyocytes: altered gene expression for detoxification enzymes

The effect of Schisandra fructus extract (SFE) on doxorubicin (Dox)-induced cardiotoxicity was investigated in H9c2 cardiomyocytes. Dox, which is an antineoplastic drug known to induce cardiomyopathy possibly through production of reactive oxygen species, induced significant cytotoxicity, intracellular reactive oxygen species (ROS), and lipid peroxidation. SFE treatment significantly increased cell survival up to 25%, inhibited intracellular ROS production in a time- and dose-dependent manner, and inhibited lipid peroxidation induced by Dox. In addition, SFE treatment induced expression of cellular glutathione S-transferases (GSTs), which function in the detoxification of xenobiotics, and endogenous toxicants including lipid peoxides. Analyses of 31,100 genes using Affymetrix cDNA microarrays showed that SFE treatment up-regulated expression of genes involved in glutathione metabolism and detoxification [GST theta 1, mu 1, and alpha type 2, heme oxygenase 1 (HO-1), and microsomal epoxide hydrolase (mEH)] and energy metabolism [carnitine palmitoyltransferase-1 (CPT-1), transaldolase, and transketolase]. These data indicated that SFE might increase the resistance to cardiac cell injury by Dox, at least partly, together with altering gene expression, especially induction of phase II detoxification enzymes.     

Stereochemical Features of Glutathione-dependent Enzymes in the Sphingobium sp. Strain SYK-6 β-Aryl Etherase Pathway

Glutathione-dependent enzymes play important protective, repair, or metabolic roles in cells. In particular, enzymes in the glutathione S-transferase (GST) superfamily function in stress responses, defense systems, or xenobiotic detoxification. Here, we identify novel features of bacterial GSTs that cleave β-aryl ether bonds typically found in plant lignin. Our data reveal several original features of the reaction cycle of these GSTs, including stereospecific substrate recognition and stereoselective formation of β-S-thioether linkages. Products of recombinant GSTs (LigE, LigP, and LigF) are β-S-glutathionyl-α-keto-thioethers that are degraded by a β-S-thioetherase (LigG). All three Lig GSTs produced the ketone product (β-S-glutathionyl-α-veratrylethanone) from an achiral side chain-truncated model substrate (β-guaiacyl-α-veratrylethanone). However, when β-etherase assays were conducted with a racemic model substrate, β-guaiacyl-α-veratrylglycerone, LigE- or LigP-catalyzed reactions yielded only one of two potential product (β-S-glutathionyl-α-veratrylglycerone) epimers, whereas the other diastereomer (differing in configuration at the β-position (i.e. its β-epimer)) was produced only in the LigF-catalyzed reaction. Thus, β-etherase catalysis causes stereochemical inversion of the chiral center, converting a β(R)-substrate to a β(S)-product (LigE and LigP), and a β(S)-substrate to a β(R)-product (LigF). Further, LigG catalyzed glutathione-dependent β-S-thioether cleavage with β-S-glutathionyl-α-veratrylethanone and with β(R)-configured β-S-glutathionyl-α-veratrylglycerone but exhibited no or significantly reduced β-S-thioether-cleaving activity with the β(S)-epimer, demonstrating that LigG is a stereospecific β-thioetherase. We therefore propose that multiple Lig enzymes are needed in this β-aryl etherase pathway in order to cleave the racemic β-ether linkages that are present in the backbone of the lignin polymer.