Evangelia Chronopoulou

Glutathione Transferases: Emerging Multidisciplinary Tools in Red and Green Biotechnology

Cytosolic glutathione transferases (GSTs) are a diverse family of enzymes involved in a wide range of biological processes, many of which involve the conjugation of the tripeptide glutathione (GSH) to an electrophilic substrate. Detailed studies of GSTs are justified because of the considerable interest of these enzymes in medicine, agriculture and analytical biotechnology. For example, in medicine, GSTs are explored as molecular targets for the design of new anticancer drugs as a plausible means to sensitize drug-resistant tumors that overexpress GSTs. In agriculture, GSTs are exploited in the development of transgenic plants with increased resistance to biotic and abiotic stresses. Recently, selected isoenzymes of GSTs have found successful applications in the development of enzyme biosensors for the direct monitoring of environmental pollutants, such as herbicides and insecticides. This review article summarizes recent representative patents related to GSTs and their applications in biotechnology.

Cloning and Characterization of a Biotic-Stress-Inducible Glutathione Transferase from Phaseolus vulgaris

Glutathione transferases (GSTs, EC 2.5.1.18) are ubiquitous proteins in plants that play important roles in stress tolerance and in the detoxification of toxic chemicals and metabolites. In this study, we systematically examined the catalytic diversification of a GST isoenzyme from Phaseolus vulgaris (PvGST) which is induced under biotic stress treatment (Uromyces appendiculatus infection). The full-length cDNA of this GST isoenzyme (termed PvGSTU3-3) with complete open reading frame, was isolated using RACE-RT and showed that the deduced amino acid sequence shares high homology with the tau class plant GSTs. PvGSTU3-3 catalyzes several different reactions and exhibits wide substrate specificity. Of particular importance is the finding that the enzyme shows high antioxidant catalytic function and acts as hydroperoxidase, thioltransferase, and dehydroascorbate reductase. In addition, its Km for GSH is about five to ten times lower compared to other plant GSTs, suggesting that PvGSTU3-3 is able to perform efficient catalysis under conditions where the concentration of reduced glutathione is low (e.g., oxidative stress). Its ability to conjugate GSH with isothiocyanates may provide an additional role for this enzyme to act as a regulator of the released isothiocyanates from glucosinolates as a response of biotic stress. Molecular modeling showed that PvGSTU3-3 shares the same overall fold and structural organization with other plant cytosolic GSTs, with major differences at their hydrophobic binding sites (H-sites) and some differences at the level of C-terminal domain and the linker between the C- and N-terminal domains. PvGSTU3-3, in general, exhibits restricted ability to bind xenobiotics in a nonsubstrate manner, suggesting that the biological role of PvGSTU3-3, is restricted mainly to the catalytic function. Our findings highlight the functional and catalytic diversity of plant GSTs and demonstrate their pivotal role for addressing biotic stresses in Phaseolus vulgaris.

Catalytic and structural diversity of the fluazifop-inducible glutathione transferases from Phaseolus vulgaris

Plant glutathione transferases (GSTs) comprise a large family of inducible enzymes that play important roles in stress tolerance and herbicide detoxification. Treatment of Phaseolus vulgaris leaves with the aryloxyphenoxypropionic herbicide fluazifop-p-butyl resulted in induction of GST activities. Three inducible GST isoenzymes were identified and separated by affinity chromatography. Their full-length cDNAs with complete open reading frame were isolated using RACE-RT and information from N-terminal amino acid sequences. Analysis of the cDNA clones showed that the deduced amino acid sequences share high homology with GSTs that belong to phi and tau classes. The three isoenzymes were expressed in E. coli and their substrate specificity was determined towards 20 different substrates. The results showed that the fluazifop-inducible glutathione transferases from P. vulgaris (PvGSTs) catalyze a broad range of reactions and exhibit quite varied substrate specificity. Molecular modeling and structural analysis was used to identify key structural characteristics and to provide insights into the substrate specificity and the catalytic mechanism of these enzymes. These results provide new insights into catalytic and structural diversity of GSTs and the detoxifying mechanism used by P. vulgaris.

Site‐saturation Mutagenesis: A Powerful Tool for Structure‐Based Design of Combinatorial Mutation Libraries

This unit describes a method for site‐saturation mutagenesis (SSM) using PCR amplification with degenerate synthetic oligonucleotides as primers. SSM allows the substitution of predetermined protein sites against all twenty possible amino acids at once. Therefore, SSM is a powerful approach in protein engineering to characterize structure‐function relationships, as well as to create improved protein variants. The procedure accepts double‐stranded plasmid isolated from the dam+ E. coli strain. The procedure is simple, fast, efficient, and eliminates time‐consuming subcloning and ligation steps. Curr. Protoc. Protein Sci. 63:26.6.1‐26.6.10.

Catalytic features and crystal structure of a tau class glutathione transferase from Glycine max specifically upregulated in response to soybean mosaic virus infections

The plant tau class glutathione transferases (GSTs) play important roles in biotic and abiotic stress tolerance in crops and weeds. In this study, we systematically examined the catalytic and structural features of a GST isoenzyme from Glycine max (GmGSTU10-10). GmGSTU10-10 is a unique isoenzyme in soybean that is specifically expressed in response to biotic stress caused by soybean mosaic virus (SMV) infections. GmGSTU10-10 was cloned, expressed in Escherichia coli, purified and characterized. The results showed that GmGSTU10-10 catalyzes several different reactions and exhibits wide substrate specificity. Of particular importance is the finding that the enzyme shows high antioxidant catalytic function and acts as hydroperoxidase. In addition, its Km for GSH is significantly lower, compared to other plant GSTs, suggesting that GmGSTU10-10 is able to perform efficient catalysis under conditions where the concentration of reduced glutathione is low (e.g. oxidative stress). The crystal structure of GmGSTU10-10 was solved by molecular replacement at 1.6Å resolution in complex with glutathione sulfenic acid (GSOH). Structural analysis showed that GmGSTU10-10 shares the same overall fold and domain organization as other plant cytosolic GSTs; however, major variations were identified in helix H9 and the upper part of helix H4 that affect the size of the active site pockets, substrate recognition and the catalytic mechanism. The results of the present study provide new information into GST diversity and give further insights into the complex regulation and enzymatic functions of this plant gene superfamily.

Plant glutathione transferase-mediated stress tolerance: functions and biotechnological applications

Plant glutathione transferases (EC 2.5.1.18, GSTs) are an ancient, multimember and diverse enzyme class. Plant GSTs have diverse roles in plant development, endogenous metabolism, stress tolerance, and xenobiotic detoxification. Their study embodies both fundamental aspects and agricultural interest, because of their ability to confer tolerance against biotic and abiotic stresses and to detoxify herbicides. Here we review the biotechnological applications of GSTs towards developing plants that are resistant to biotic and abiotic stresses. We integrate recent discoveries, highlight, and critically discuss the underlying biochemical and molecular pathways involved. We elaborate that the functions of GSTs in abiotic and biotic stress adaptation are potentially a result of both catalytic and non-catalytic functions. These include conjugation of reactive electrophile species with glutathione and the modulation of cellular redox status, biosynthesis, binding, and transport of secondary metabolites and hormones. Their major universal functions under stress underline the potential in developing climate-resilient cultivars through a combination of molecular and conventional breeding programs. We propose that future GST engineering efforts through rational and combinatorial approaches, would lead to the design of improved isoenzymes with purpose-designed catalytic activities and novel functional properties. Concurrent GST–GSH metabolic engineering can incrementally increase the effectiveness of GST biotechnological deployment.

Characterization and functional analysis of a recombinant tau class glutathione transferase GmGSTU2-2 from Glycine max.

The plant tau class glutathione transferases (GSTs) perform diverse catalytic as well as non-catalytic roles in detoxification of xenobiotics, prevention of oxidative damage and endogenous metabolism. In the present work, the tau class isoenzyme GSTU2-2 from Glycine max (GmGSTU2-2) was characterized. Gene expression analysis of GmGSTU2 suggested a highly specific and selective induction pattern to osmotic stresses, indicating that gene expression is controlled by a specific mechanism. Purified, recombinant GmGSTU2-2 was shown to exhibit wide-range specificity towards xenobiotic compounds and ligand-binding properties, suggesting that the isoenzyme could provide catalytic flexibility in numerous metabolic conditions. Homology modeling and phylogenetic analysis suggested that the catalytic and ligand binding sites of GmGSTU2-2 are well conserved compared to other tau class GSTs. Structural analysis identified key amino acid residues in the hydrophobic binding site and provided insights into the substrate specificity of this enzyme. The results established that GmGSTU2-2 participates in a broad network of catalytic and regulatory functions involved in the plant stress response.

Directed evolution of glutathione transferases towards a selective glutathione-binding site and improved oxidative stability.

BACKGROUND Glutathione transferases (GSTs) are a family of detoxification enzymes that catalyze the conjugation of glutathione (GSH) to electrophilic compounds. METHODS A library of alpha class GSTs was constructed by DNA shuffling using the DNA encoding the human glutathione transferase A1-1 (hGSTA1-1) and the rat glutathione transferase A1-1 (rGSTA1-1). RESULTS Activity screening of the library allowed the selection of a chimeric enzyme variant (GSTD4) that displayed high affinity towards GSH and GSH-Sepharose affinity adsorbent, higher kcat/Km and improved thermal stability, compared to the parent enzymes. The crystal structures of the GSTD4 enzyme in free form and in complex with GSH were determined to 1.6Å and 2.3Å resolution, respectively. Analysis of the GSTD4 structure showed subtle conformational changes in the GSH-binding site and in electron-sharing network that may contribute to the increased GSH affinity. The shuffled variant GSTD4 was further optimized for improved oxidative stability employing site-saturation mutagenesis. The Cys112Ser mutation confers optimal oxidative stability and kinetic properties in the GSTD4 enzyme. CONCLUSIONS DNA shuffling allowed the creation of a chimeric enzyme variant with improved properties, compared to the parent enzymes. X-ray crystallography shed light on how recombination of a specific segment from homologous GSTA1-1 together with point mutations gives rise to a new functionally competent enzyme with improved binding, catalytic properties and stability. GENERAL SIGNIFICANCE Such an engineered GST would be useful in biotechnology as affinity tool in affinity chromatography as well as a biocatalytic matrix for the construction of biochips or enzyme biosensors.

Plant Glutathione Transferases: Structure, Antioxidant Catalytic Function and in planta Protective Role in Biotic and Abiotic Stress

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Structure and Antioxidant Catalytic Function of Plant Glutathione Transferases

Plant cytosolic glutathione transferases (GSTs) are an ancient enzyme superfamily with multiple and diverse functions which are important in counteracting biotic and abiotic stress. GSTs play an important role in catalyzing the conjugation of xenobiotics and endogenous electrophilic compounds with glutathione (GSH), such as pesticides, chemical carcinogens, environmental pollutants, which leads to their detoxification. GSTs not only catalyze detoxification reactions but they are also involved in GSH-dependent isomerization reactions, in GSH-dependent reduction of organic hydroper- oxides formed during oxidative stress, biosynthesis of sulfur-containing secondary metabolites, and exhibit thioltrans- ferase and dehydroascorbate reductase activity. This review focuses on plant GSTs, and attempts to give an overview of the new insights into the catalytic function and structural biology of these enzymes.