Zeta Class Glutathione S-transferases Research Articles

Zeta class glutathione S-transferase is involved in phoxim tolerance and is potentially regulated by the transcription factor CncC in Agrotis ipsilon (Lepidoptera: Noctuidae)

The black cutworm, Agrotis ipsilon (Lepidoptera: Noctuidae), is an important agricultural pest. Phoxim is an organophosphate insecticide that has been widely used to control A. ipsilon. The extensive application of phoxim has resulted in a reduction in phoxim susceptibility in A. ipsilon. However, the molecular mechanisms underlying phoxim tolerance in A. ipsilon remain unclear. In this work, we report the involvement of AiGSTz1, a zeta class glutathione S-transferase, in phoxim tolerance in A. ipsilon. Exposure to a sublethal concentration (LC50) of phoxim dramatically upregulated the transcription level of the AiGSTz1 gene in A. ipsilon larvae, and this upregulation might be caused by phoxim-induced oxidative stress. The recombinant AiGSTz1 protein expressed in Escherichia coli was able to metabolize phoxim. Furthermore, AiGSTz1 displayed antioxidant activity to protect against oxidative stress. Knockdown of AiGSTz1 by RNA interference significantly increased the mortality rate of A. ipsilon larvae in response to phoxim. In addition, the transcription factor AiCncC can bind to the cap ‘n’ collar isoform C: muscle aponeurosis fibromatosis (CncC:Maf) binding site in the putative promoter of the AiGSTz1 gene. Silencing of AiCncC resulted in a dramatic downregulation of AiGSTz1. These results indicated that AiGSTz1 is involved in phoxim tolerance and is potentially regulated by AiCncC. These findings provide valuable insights into the defense mechanisms used by A. ipsilon against phoxim.

拟南芥AtGSTZ1基因启动子与GUS重组载体的构建及转化

拟南芥AtGSTZ1基因启动子与GUS重组载体的构建及转化

Functional Role of Tyr12 in the Catalytic Activity of Novel Zeta-like Glutathione S-transferase from Acidovorax sp. KKS102.

Glutathione S-transferases (GSTs) are a family of enzymes that function in the detoxification of variety of electrophilic substrates. In the present work, we report a novel zeta-like GST (designated as KKSG9) from the biphenyl/polychlorobiphenyl degrading organism Acidovorax sp. KKS102. KKSG9 possessed low sequence similarity but similar biochemical properties to zeta class GSTs. Functional analysis showed that the enzyme exhibits wider substrate specificity compared to most zeta class GSTs by reacting with 1-chloro-2,4-dinitrobenzene (CDNB), p-nitrobenzyl chloride (NBC), ethacrynic acid (EA), hydrogen peroxide, and cumene hydroperoxide. The enzyme also displayed dehalogenation function against dichloroacetate, permethrin, and dieldrin. The functional role of Tyr12 was also investigated by site-directed mutagenesis. The mutant (Y12C) displayed low catalytic activity and dehalogenation function against all the substrates when compared with the wild type. Kinetic analysis using NBC and GSH as substrates showed that the mutant (Y12C) displayed a higher affinity for NBC when compared with the wild type, however, no significant change in GSH affinity was observed. These findings suggest that the presence of tyrosine residue in the motif might represent an evolutionary trend toward improving the catalytic activity of the enzyme. The enzyme as well could be useful in the bioremediation of various types of organochlorine pollutants.

A combined biochemical and computational studies of the rho-class glutathione s-transferase sll1545 of Synechocystis PCC 6803

Peroxides are one of the most important radicals that cause oxidative stress. Certain Glutathione S-transferases (GSTs) have been reported to show peroxidase activity. We report a novel peroxidase activity of Synechocystis GST- sll1545. The recombinant protein was purified to homogeneity and characterized. Low Km (0.109μM) and high Vmax (0.663μmolmin−1) values suggest a high preference of sll1545 for cumenehydroperoxide. Disc inhibition assay confirmed the ability of the enzyme to protect cells against peroxide-induced damage. sll1545 has very low sequence and structural similarity with theta and alpha class GSTs that exhibit glutathione-dependent peroxidase activity. Recent data from our laboratory shows that sll1545 is also strongly active against dichloroacetate (DCA), which is a characteristic of zeta class GST. Interestingly, sll1545 shows less than 20% sequence identity with zeta class GST. Molecular dynamic simulation results show that sll1545 was much more structurally different from alpha/theta classes. Our results suggest that sll1545 shows structural variation from zeta, theta/alpha classes of GSTs but have related enzymatic activity. Phylogenetic analysis reveal that sll1545 is evolutionally very distinct from the known GSTs. Overall, the data suggest that Synechocystis sll1545 does not belong to any known GST class and represent a novel GST class, which we have named rho.

Discovery of Organophosphate Resistance-Related Genes Associated With Well-known Resistance Mechanisms of Plutella xylostella (L.) (Lepidoptera: Plutellidae) by RNA-Seq.

Pesticide resistance poses many challenges for pest control, particularly for destructive pests such as diamondback moths ( Plutella xylostella ). Organophosphates have been used in the field since the 1950s, leading to selection for resistance-related gene variants and the development of resistance to new insecticides in the diamondback moth. Identifying actual and potential genes involved in resistance could offer solutions for control. This study established resistant diamondback moth strains from two different collections using mevinphos. Two sets of transcriptome sequencing (RNA-Seq) data were generated for pairs of mevinphos-resistant versus susceptible (wild-type) strains. One susceptible strain containing 14 giga base pairs was assembled into a reference-based assembly using published scaffold sequences as reference. Differential expression data between resistant and susceptible strains revealed 944 transcripts (803 with annotations) showing upregulation and 427 transcripts (150 with annotations) showing downregulation. Around 6.8% of the differential expression transcripts (65) could be categorized as associated with well-known resistance mechanisms such as penetration, detoxification, and behavior response; of these 65 transcripts, 38 showed upregulation, and 12 relating to penetration were upregulated when the transcripts of 19 cytochrome P450s, 2 zeta-class glutathione S-transferases, and 4 ATP-binding cassette transporters showed upregulation. In addition, 11 groups of transcripts related to olfactory perception appeared to be downregulated in trade-off situations. Quantitative polymerase chain reaction expression results were consistent with RNA-Seq data. Possible roles of these differentially expressed genes in resistance mechanisms are discussed in this study.

De novo analysis of the Tenebrio molitor (Coleoptera: Tenebrionidae) transcriptome and identification of putative glutathione S-transferase genes

We performed the Illumina sequencing of the yellow mealworm beetle, Tenebrio molitor L., a global stored grain pest and a model for insect physiological studies, and analyzed the transcriptome dataset. We obtained 82,001,576 clean reads that were assembled into 31,785 unigenes with a mean size of 1,022 bp. Among these unigenes, 16,025 (50.41 %) showed significant similarity (E value <10−5) to known proteins in the NCBI non-redundant database. Gene ontology, Cluster of Orthologous Groups and Kyoto Encyclopedia of Genes and Genomes analyses were used to classify functions of the unigenes. We identified 27 putative glutathione S-transferase (GST) genes, including 2 delta, 13 epsilon, 1 omega, 5 sigma, 1 theta and 1 zeta class GSTs, and 2 microsomal GSTs and 2 “unclassified” GSTs. Quantitative reverse transcription-PCR assay showed that 2 and 3 GST genes were mainly expressed in larval and pupal stages, respectively; while 10 genes were mainly expressed in the adult stage. Tissue-specific expression analysis found that, among the 27 GST genes, 8 were expressed predominately in the midgut, 13 were mainly detected in Malpighian tubules, and only one gene specifically distributed in the fat body. This work provides the first insights into the T. molitor GSTs at the molecular level: the results may assist in a functional study for the GSTs in this insect species.

The identification and oxidative stress response of a zeta class glutathione S-transferase (GSTZ1) gene from Apis cerana cerana

Glutathione-S-transferases (GSTs) play an important role in protecting organisms against the toxicity of reactive oxygen species (ROS). However, no information is available for GSTs in the Chinese honey bee (Apis cerana cerana). In this study, we isolated and characterized a zeta class GST gene (AccGSTZ1) from the Chinese honey bee. This gene is present in a single copy and harbors five exons. The deduced amino acid sequence of AccGSTZ1 shared high sequence identity with homologous proteins and contained the highly conserved features of this gene family. The temporal and spatial expression profiles of AccGSTZ1 showed that AccGSTZ1 was highly expressed in fourth instar larvae during development, and the mRNA level of AccGSTZ1 was higher in the epidermis than that in other tissues. The expression pattern under oxidative stress revealed that AccGSTZ1 transcription was significantly upregulated by external factors, such as temperature challenges and H2O2 treatment. The characterization of the purified protein revealed that AccGSTZ1 had low glutathione-conjugating activity, but the recombinant AccGSTZ1 protein displayed high antioxidant activity under oxidative stress. These data suggest that AccGSTZ1 is an oxidative stress-inducible antioxidant enzyme that plays an important role in the protection against oxidative stress and may be of critical importance for the survival of the honey bees.

Molecular cloning and expression analysis of a zeta-class glutathione S-transferase gene in sugarcane

Glutathione S-transferases (GSTs) play an important role in stress tolerance in plants. This is the first report of cloning and characterization of a zeta-class GST gene in sugarcane (GenBank Accession number: GQ246461). Sequence analysis showed that the cDNA sequence of Sc-GST gene was 829 bp, contained a 621 bp open reading frame (ORF), the 5’ untranslated region (UTR) of 65 bp and 3’UTR of 143 bp, plus the typical AATAA region and poly (A) tail. It encoded the 206 amino acid residues with a molecular mass of 23.1 KD and isoelectric point of 6.10. Protein domain prediction and multiple sequence alignment demonstrated that the conserved domain in Sc-GST at N-terminus was SSCXXRXRIA, while that at C-terminus was quebec platelet disorder (QPD), both of which were specific for zeta-type GST in eukaryotes. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis and enzyme activity assay indicated that the prokaryotic expression product was a fusion protein with a molecular weight of about 30 KD, which also possessed GST enzyme activity. It was revealed in real-time quantitative polymerase chain reaction (qPCR) analysis that the Sc-GST gene had induced expression under H 2 O 2 and Ustilago scitaminea stresses, while it was inhibited and then induced by salicylic acid (SA) stress, suggesting that it is a type of stress-tolerant gene playing a certain role in sugarcane resistance response. Key words: Saccharum officinarum, glutathione S-transferase, homology, prokaryotic expression, real-time quantitative PCR.

Molecular and biochemical characterization of a Zeta-class glutathione S-transferase of the silkmoth

A cDNA encoding Zeta-class GST of the silkmoth, Bombyx mori (bmGSTZ), was cloned by a reverse transcriptase-polymerase chain reaction. The resulting clone was sequenced and deduced for amino acid sequence, which revealed 45–50% identities to Zeta-class GSTs from other organisms. A recombinant protein (rbmGSTZ) was functionally overexpressed in Bombyx mori cells in a soluble form and purified to homogeneity. rbmGSTZ was able to catalyze the biotranslation of glutathione with dichloroacetic acid. We found that the present GST-catalyzed dechlorination of permethrin and distributed abundantly in silkmoth strain exhibiting permethrin resistance. Our results suggest that bmGSTZ could contribute to permethrin resistance in lepidopteran.

Characterization of Ser73 in Arabidopsis thaliana Glutathione S-transferase zeta class

Glutathione S-transferases (GSTs) are ubiquitous detoxifying superfamily enzymes. The zeta class GST from Arabidopsis thaliana ( AtGSTZ) can efficiently degrade dichloroacetic acid (DCA), which is a common carcinogenic contaminant in drinking water. Ser73 in AtGSTZ is a conserved residue at Glutathione binding site (G-site). Compared with the equivalent residues in other GSTs, the catalytic and structural properties of Ser73 were poorly investigated. In this article, site-saturation mutagenesis was performed to characterize the detailed role of Ser73. The DCA dechlorinating (DCA-DC) activity showed that most of the mutants had less than 3% of the wild-type activity, except S73T and S73A showing 43.48% and 21.62% of the wild-type activity, respectively, indicating that position 73 in AtGSTZ showed low mutational substitutability. Kinetic experiments revealed that mutants S73T, S73A, and S73G showed low binding affinity and catalytic efficiency toward DCA, 1.8-, 3.1-, and 10.7-fold increases in K m DCA values and 4.0-, 9.6-, and 34.1-fold decreases in K cat- DCA/ K m DCA values, respectively, compared to the wild type. Thermostability and refolding experiments showed that the wild type maintained more thermostability and recovered activity. These results demonstrated the important role of Ser73 in catalytic activity and structural stability of the enzyme. Such properties of Ser73 could be particularly crucial to the molecular evolution of AtGSTZ and might, therefore, help explain why Ser73 is conserved in all GSTs. This conclusion might provide insights into the directed evolution of the DCA-DC activity of AtGSTZ.

Fine mapping of the qCTS12 locus, a major QTL for seedling cold tolerance in rice

The temperate japonica rice cultivar M202 is the predominant variety grown in California due to its tolerance to low temperature stress, good grain quality and high yield. Earlier analysis of a recombinant inbred line mapping population derived from a cross between M202 and IR50, an indica cultivar that is highly sensitive to cold stress, resulted in the identification of a number of QTL conferring tolerance to cold-induced wilting and necrosis. A major QTL, qCTS12, located on the short arm of chromosome 12, contributes over 40% of the phenotypic variance. To identify the gene(s) underlying qCTS12, we have undertaken the fine mapping of this locus. Saturating the short arm of chromosome 12 with microsatellite markers revealed that qCTS12 is closest to RM7003. Using RM5746 and RM3103, which are immediately outside of RM7003, we screened 1,954 F(5)-F(10) lines to find recombinants in the qCTS12 region. Additional microsatellite markers were identified from publicly available genomic sequence and used to fine map qCTS12 to a region of approximately 87 kb located on the BAC clone OSJNBb0071I17. This region contains ten open reading frames (ORFs) consisting of five hypothetical and expressed proteins of unknown function, a transposon protein, a putative NBS-LRR disease resistance protein, two zeta class glutathione S-transferases (OsGSTZ1 and OsGSTZ2), and a DAHP synthetase. Further fine mapping with markers developed from the ORFs delimited the QTL to a region of about 55 kb. The most likely candidates for the gene(s) underlying qCTS12 are OsGSTZ1 and OsGSTZ2.

Genomic structure and differential expression of two tandem-arranged GSTZ genes in rice

Glutathione S-transferases (GSTs) are scavenging enzymes that detoxify cellular xenobiotics and toxins by catalyzing the conjugation of these substrates with a tripeptide glutathione. GSTs are classified depending on gene organization and sequence similarity. The sequence analysis of genomic DNA for zeta class GST ( GSTZ) locus in rice indicated that two homologous GSTZ genes lay in a tandem orientation with a short (0.4 kb) intergenic spacer. The upstream OsGSTZ1 and downstream OsGSTZ2 spanned 3.5 and 3.2 kb with nine coding exons, respectively. The transcript of OsGSTZ1 had a long 3′ untranslated region (3′ UTR) that was mostly encoded by a 10th noncoding exon, whereas OsGSTZ2 mRNA contained a long 5′ UTR. Northern blot analysis showed that OsGSTZ1/2 messages were strongly expressed in leaf blades, while transcripts from roots were low level. Because OsGSTZ1/2 messages in leaf tissues were strongly induced only by water treatment, it was difficult to assay for the induction of OsGSTZ1/2 transcripts by various stress treatments. Thus, using rice culture cells, we analyzed the respective responses of OsGSTZ1 and OsGSTZ2 genes against various treatments by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The results showed that OsGSTZ1 was expressed at a level ca. 1000-fold higher than OsGSTZ2 in suspension cells without stress treatment. OsGSTZ1 was expressed constitutively under various stress conditions. In contrast, the expression of OsGSTZ2 gene was strongly enhanced to 30-fold by treatment with jasmonic acid. These observations suggested that the expression of OsGSTZ1 and OsGSTZ2 genes are differentially regulated in the culture cell of rice.

Abiotic stress alters transcript profiles and activity of glutathione S-transferase, glutathione peroxidase, and glutathione reductase in Euphorbia esula.

Glutathione S-transferase (GST), glutathione peroxidase (GPX), and glutathione reductase (GR) are enzymes that utilize glutathione to play an important role in plant defense mechanisms. In leafy spurge (Euphorbia esula L.), transcript and activity profiles for these enzymes are differentially influenced in tissue exposed to xenobiotic (diclofop-methyl) and environmental stress (cold and drought). Five different EeGST cDNA (including phi, tau, theta, and zeta class GSTs), one EeGPX cDNA, and one EeGR cDNA showed differential expression patterns in leafy spurge plants exposed to diclofop-methyl-, cold- and drought-stress. Tissue treated with diclofop-methyl also had increased GST, GPX and GR activities that were preceded or paralleled by increased gene expression. Transcript profiles resulting from drought-stressed plants were similar to transcript profiles from diclofop-methyl-treated plants but not cold-stressed plants. GPX activity in leafy spurge protein extracts was not bound to either S-hexylglutathione- or glutathione-agarose columns but instead co-migrated with fractions of GST activity that also were not bound by affinity chromatography. Fractions of GST proteins that were bound to S-hexylglutathione revealed that increased GST activity in diclofop-methyl-treated tissue could be identified as phi- and tau-type GSTs.

Disruption of GSTZ1 Gene by Large Genetic Alteration in Oryza glaberrima

After the completion of the genome sequencing project of common rice (Oryza sativa L.), comparative genomic studies between rice and related species became important to reveal the function of each gene. The rice genome contains two copies of the gene encoding zeta class glutathione S-transferase (GSTZ) that is reported to be the enzyme in the catabolic pathway of tyrosine and phenylalanine. Two GSTZ genes of O. sativa, OsGSTZ1 and OsGSTZ2, display a tandem arrangement. Upstream OsGSTZ1 gene is constitutively expressed, whereas the downstream OsGSTZ2 gene is inducible by stresses. We analyzed the expression of the GSTZ gene in the African cultivated species O. glaberrima and wild species O. longistaminata by using RT-PCR. The results showed that both GSTZ1 and GSTZ2 genes were expressed in O. longistaminata, whereas only the cDNA fragment of downstream GSTZ2 was detected in O. glaberrima. Thus, the genomic sequence of the GSTZ1 locus of O. glaberrima was determined by PCR genomic walking. Sequence comparison between O. sativa and O. glaberrima revealed that the genomic sequence upstream from the eighth intron of the OgGSTZ1 gene was highly homologous, in inverted orientation, to a BAC clone, over 1-Mb apart from the OsGSTZ1 gene in O. sativa. This result suggested that the OgGSTZ1 gene was disrupted by a large rearrangement or inversion. The genetic alteration was also observed in several lines of O. glaberrima and its ancestral wild species O. barthii. The loss of the OgGSTZ1 gene in O. glaberrima is important, as the expression of the gene is 12-fold higher than that of OsGSTZ2 in O. sativa and since O. sativa and O. glaberrima differ in several such as seed yield and annual/perennial habitat that might be related to the translocation of nitrogen metabolites from leaves to seeds.

The structure of a zeta class glutathione S-transferase from Arabidopsis thaliana: characterisation of a GST with novel active-site architecture and a putative role in tyrosine catabolism

The cis-trans isomerisation of maleylacetoacetate to fumarylacetoacetate is the penultimate step in the tyrosine/phenylalanine catabolic pathway and has recently been shown to be catalysed by glutathione S-transferase enzymes belonging to the zeta class. Given this primary metabolic role it is unsurprising that zeta class glutathione S-transferases are well conserved over a considerable period of evolution, being found in vertebrates, plants, insects and fungi. The structure of this glutathione S-transferase, cloned from Arabidopsis thaliana, has been solved by single isomorphous replacement with anomalous scattering and refined to a final crystallographic R-factor of 19.6% using data from 25.0 Å to 1.65 Å. The zeta class enzyme adopts the canonical glutathione S-transferase fold and forms a homodimer with each subunit consisting of 221 residues. In agreement with structures of glutathione S-transferases from the theta and phi classes, a serine residue (Ser17) is present in the active site, at a position that would allow it to stabilise the thiolate anion of glutathione. Site-directed mutagenesis of this residue confirms its importance in catalysis. In addition, the role of a highly conserved cysteine residue (Cys19) present in the active site of the zeta class glutathione S-transferase enzymes is discussed.

Isolation of a Zeta class wheat glutathione S-transferase gene

A new Zeta class glutathione S-transferases (GST) gene, pGST, has been cloned from wheat for the first time by the differential display PCR (DD-PCR) method. The genomic sequence of pGST, TA-GSTZ1, contains nine exons that encode a polypeptide of 213 amino acids and eight introns. The deduced amino acid sequence of TA-GSTZ1 as well as the exon:intron placement are more similar to the GSTs of the Zeta class than to the two wheat GSTs reported earlier. The pGST cDNA gene product expressed in Escherichia coli and purified by affinity chromatography showed typical Zeta class GST and glutathione peroxidase activities. Sequence polymorphism in the 3′ untranslated region (UTR) of TA-GSTZ1 gene in wheat has been discovered. In this study, an 89 bp sequence is present in the 3′ UTR of TA-GSTZ1gene in 16 wheat cultivars but absent in the other five. Although the biological importance of this polymorphism is unknown, it can be useful as a genetic marker in wheat breeding.