Catalytic properties of glutathione-binding residues in a tau class glutathione transferase (PtGSTU1) from Pinus tabulaeformis

Glutathione transferases (GSTs) play important roles in stress tolerance and detoxification in plants. However, there is extremely little information on the molecular characteristics of GSTs in gymnosperms. In a previous study, we cloned a tau class GST (PtGSTU1) from a gymnosperm (Pinus tabulaeformis) for the first time. Based on the N-terminal amino acid sequence identity to the available crystal structures of plant tau GSTs, Ser13, Lys40, Ile54, Glu66 and Ser67 of PtGSTU1 were proposed as glutathione-binding (G-site) residues. The importance of Ser13 as a G-site residue was investigated previously. The functions of Lys40, Ile54, Glu66 and Ser67 of PtGSTU1 are examined in this study through site-directed mutagenesis. Enzyme assays and thermal stability measurements on the purified recombinant PtGSTU1 showed that substitution at each of these sites significantly affects the enzyme's substrate specificity and affinity for GSH, and these residues are essential for maintaining the stability of PtGSTU1. The results of protein expression and refolding analyses suggest that Ile54 is involved in the protein folding process. The findings demonstrate that the aforementioned residues are critical components of active sites that contribute to the enzyme's catalytic activity and structural stability.     

A conserved hydrogen-bond network stabilizes the structure of Beta class glutathione S-transferases

We identified a network of hydrogen bonds that is conserved in the structures of bacterial Beta class glutathione S-transferases (GSTs). It is formed by three residues: a serine, a histidine and a glutamate, together with a water molecule that links the serine with the histidine. This network connects the first helix of the N-terminal glutaredoxin-like domain with the last helix of the C-terminal GST-specific all helical domain. Here we show that substitution of Ochrobactrum anthropi GST His15 and Glu198 with alanine greatly compromises the catalytic efficiency of the enzyme, even though none of these residues takes part to the enzyme active site. Thermal and chemical denaturation experiments point to a role for this network in global structure stabilization. Furthermore, we show that OaGST structure looses compactness at alkanine pHs and that this behavior may be ascribed to partial disruption of the H-bond network, pointing to an important role in zippering the N-terminal and C-terminal domains of the protein.     

Crystal structure of a theta-class glutathione transferase.

Glutathione S-transferases (GSTs) are a family of enzymes involved in the cellular detoxification of xenotoxins. Cytosolic GSTs have been grouped into four evolutionary classes for which there are representative crystal structures of three of them. Here we report the first crystal structure of a theta-class GST. So far, all available GST crystal structures suggest that a strictly conserved tyrosine near the N-terminus plays a critical role in the reaction mechanism and such a role has been convincingly demonstrated by site-directed mutagenesis. Surprisingly, the equivalent residue in the theta-class structure is not in the active site, but its role appears to have been replaced by either a nearby serine or by another tyrosine residue located in the C-terminal domain of the enzyme.     

Eukaryotic translation elongation factor 1γ contains a glutathione transferase domain—Study of a diverse,ancient protein super family using motif search and structural modeling

Using computer methods for multiple alignment, sequence motif search, and tertiary structure modeling, we show that eukaryotic translation elongation factor 1γ (EF1γ) contains an N-terminal domain related to class θ glutathione S-transferases (GST). GST-like proteins related to class θ comprise a large group including, in addition to typical GSTs and EF1γ, stress-induced proteins from bacteria and plants, bacterial reductive dehalogenases and β-etherases, and several uncharacterized proteins. These proteins share 2 conserved sequence motifs with GSTs of other classes (α, μ, and π). Tertiary structure modeling showed that in spite of the relatively low sequence similarity, the GST-related domain of EF1γ is likely to form a fold very similar to that in the known structures of class α, μ, and π GSTs. One of the conserved motifs is implicated in glutathione binding, whereas the other motif probably is involved in maintaining the proper conformation of the GST domain. We predict that the GST-like domain in EF1γ is enzymatically active and that to exhibit GST activity, EF1γ has to form homodimers. The GST activity may be involved in the regulation of the assembly of multisubunit complexes containing EF1 and aminoacyl-tRNA synthetases by shifting the balance between glutathione, disulfide glutathione, thiol groups of cysteines, and protein disulfide bonds. The GST domain is a widespread, conserved enzymatic module that may be covalently or noncovalently complexed with other proteins. Regulation of protein assembly and folding may be 1 of the functions of GST.     

The Suppressor of Killer of prune, a unique glutathione S-transferase

The prune-Killer of prune conditional dominant, lethal interaction in Drosophila was identified in the 1950s, but its mechanism remains unknown. We undertook a genetic screen for suppressors of this lethal interaction and identified a gene we named, Suppressor of Killer of prune Su(Kpn). Su(Kpn) is a unique protein with four N-terminal FLYWCH zinc-finger domains, an acidic domain and a C-terminal glutathione S-transferase (GST) domain. The GST domain of Su(Kpn) is of particular interest because GSTs are usually independent of other protein domains. While GSTs are generally thought of as detoxifying enzymes, they are also associated with cellular toxicity. We predict that the GST domain of the Su(Kpn) creates a toxic product in prune-Killer of prune flies that is lethal. The substrate of the Su(Kpn) remains unknown.     

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.     

Characterization of a Phanerochaete chrysosporium Glutathione Transferase Reveals a Novel Structural and Functional Class with Ligandin Properties

Glutathione S-transferases (GSTs) form a superfamily of multifunctional proteins with essential roles in cellular detoxification processes. A new fungal specific class of GST has been highlighted by genomic approaches. The biochemical and structural characterization of one isoform of this class in Phanerochaete chrysosporium revealed original properties. The three-dimensional structure showed a new dimerization mode and specific features by comparison with the canonical GST structure. An additional β-hairpin motif in the N-terminal domain prevents the formation of the regular GST dimer and acts as a lid, which closes upon glutathione binding. Moreover, this isoform is the first described GST that contains all secondary structural elements, including helix α4′ in the C-terminal domain, of the presumed common ancestor of cytosolic GSTs (i.e. glutaredoxin 2). A sulfate binding site has been identified close to the glutathione binding site and allows the binding of 8-anilino-1-naphtalene sulfonic acid. Competition experiments between 8-anilino-1-naphtalene sulfonic acid, which has fluorescent properties, and various molecules showed that this GST binds glutathionylated and sulfated compounds but also wood extractive molecules, such as vanillin, chloronitrobenzoic acid, hydroxyacetophenone, catechins, and aldehydes, in the glutathione pocket. This enzyme could thus function as a classical GST through the addition of glutathione mainly to phenethyl isothiocyanate, but alternatively and in a competitive way, it could also act as a ligandin of wood extractive compounds. These new structural and functional properties lead us to propose that this GST belongs to a new class that we name GSTFuA, for fungal specific GST class A.     

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.     

Extensive Functional Diversification of the Populus Glutathione S-Transferase Supergene Family

Identifying how genes and their functions evolve after duplication is central to understanding gene family radiation. In this study, we systematically examined the functional diversification of the glutathione S-transferase (GST) gene family in Populus trichocarpa by integrating phylogeny, expression, substrate specificity, and enzyme kinetic data. GSTs are ubiquitous proteins in plants that play important roles in stress tolerance and detoxification metabolism. Genome annotation identified 81 GST genes in Populus that were divided into eight classes with distinct divergence in their evolutionary rate, gene structure, expression responses to abiotic stressors, and enzymatic properties of encoded proteins. In addition, when all the functional parameters were examined, clear divergence was observed within tandem clusters and between paralogous gene pairs, suggesting that subfunctionalization has taken place among duplicate genes. The two domains of GST proteins appear to have evolved under differential selective pressures. The C-terminal domain seems to have been subject to more relaxed functional constraints or divergent directional selection, which may have allowed rapid changes in substrate specificity, affinity, and activity, while maintaining the primary function of the enzyme. Our findings shed light on mechanisms that facilitate the retention of duplicate genes, which can result in a large gene family with a broad substrate spectrum and a wide range of reactivity toward different substrates.     

Crystallographic and Functional Characterization of the Fluorodifen-inducible Glutathione Transferase from Glycine max Reveals an Active Site Topography Suited for Diphenylether Herbicides and a Novel L-site

Glutathione transferases (GSTs) from the tau class (GSTU) are unique to plants and have important roles in stress tolerance and the detoxification of herbicides in crops and weeds. A fluorodifen-induced GST isoezyme (GmGSTU4-4) belonging to the tau class was purified from Glycine max by affinity chromatography. This isoenzyme was cloned and expressed in Escherichia coli, and its structural and catalytic properties were investigated. The structure of GmGSTU4-4 was determined at 1.75 Å resolution in complex with S-(p-nitrobenzyl)-glutathione (Nb-GSH). The enzyme adopts the canonical GST fold but with a number of functionally important differences. Compared with other plant GSTs, the three-dimensional structure of GmGSTU4-4 primarily shows structural differences in the hydrphobic substrate binding site, the linker segment and the C-terminal region. The X-ray structure identifies key amino acid residues in the hydrophobic binding site (H-site) and provides insights into the substrate specificity and catalytic mechanism of the enzyme. The isoenzyme was highly active in conjugating the diphenylether herbicide fluorodifen. A possible reaction pathway involving the conjugation of glutathione with fluorodifen is described based on site-directed mutagenesis and molecular modeling studies. A serine residue (Ser13) is present in the active site, at a position that would allow it to stabilise the thiolate anion of glutathione and enhance its nucleophilicity. Tyr107 and Arg111 present in the active site are important structural moieties that modulate the catalytic efficiency and specificity of the enzyme, and participate in k_cat regulation by affecting the rate-limiting step of the catalytic reaction. A hitherto undescribed ligand-binding site (L-site) located in a surface pocket of the enzyme was also found. This site is formed by conserved residues, suggesting it may have an important functional role in the transfer and delivery of bound ligands, presumably to specific protein receptors.     

Structural and Functional Evolution of Positively Selected Sites in Pine Glutathione S-Transferase Enzyme Family

Phylogenetic analyses have identified positive selection as an important driver of protein evolution, both structural and functional. However, the lack of appropriate combined functional and structural assays has generally hindered attempts to elucidate patterns of positively selected sites and their effects on enzyme activity and substrate specificity. In this study we investigated the evolutionary divergence of the glutathione S-transferase (GST) family in Pinus tabuliformis, a pine that is widely distributed from northern to central China, including cold temperate and drought-stressed regions. GSTs play important roles in plant stress tolerance and detoxification. We cloned 44 GST genes from P. tabuliformis and found that 26 of the 44 belong to the largest (Tau) class of GSTs and are differentially expressed across tissues and developmental stages. Substitution models identified five positively selected sites in the Tau GSTs. To examine the functional significance of these positively selected sites, we applied protein structural modeling and site-directed mutagenesis. We found that four of the five positively selected sites significantly affect the enzyme activity and specificity; thus their variation broadens the GST family substrate spectrum. In addition, positive selection has mainly acted on secondary substrate binding sites or sites close to (but not directly at) the primary substrate binding site; thus their variation enables the acquisition of new catalytic functions without compromising the protein primary biochemical properties. Our study sheds light on selective aspects of the functional and structural divergence of the GST family in pine and other organisms.     

Analysis and Phylogeny of Small Heat Shock Proteins from Marine Viruses and Their Cyanobacteria Host

Small heat shock proteins (sHSPs) are oligomeric stress proteins characterized by an α-crystallin domain (ACD) surrounded by a N-terminal arm and C-terminal extension. Publications on sHSPs have reported that they exist in prokaryotes and eukaryotes but, to our knowledge, not in viruses. Here we show that sHSPs are present in some cyanophages that infect the marine unicellular cyanobacteria, Synechococcus and Prochlorococcus. These phage sHSPs contain a conserved ACD flanked by a relatively conserved N-terminal arm and a short C-terminal extension with or without the conserved C-terminal anchoring module (CAM) L-X-I/V, suggested to be implicated in the oligomerization. In addition, cyanophage sHSPs have the signature pattern, P-P-[YF]-N-[ILV]-[IV]-x(9)-[EQ], in the predicted β2 and β3 strands of the ACD. Phylogenetically, cyanophage sHSPs form a monophyletic clade closer to bacterial class A sHSPs than to cyanobacterial sHSPs. Furthermore, three sHSPs from their cellular host, Synechococcus, are phylogenetically close to plants sHSPs. Implications of evolutionary relationships between the sHSPs of cyanophages, bacterial class A, cyanobacteria, and plants are discussed.     

Biochemical and mutational analysis of EcoRII functional domains reveals evolutionary links between restriction enzymes

The archetypal Type IIE restriction endonuclease EcoRII is a dimer that has a modular structure. DNA binding studies indicate that the isolated C-terminal domain dimer has an interface that binds a single cognate DNA molecule whereas the N-terminal domain is a monomer that also binds a single copy of cognate DNA. Hence, the full-length EcoRII contains three putative DNA binding interfaces: one at the C-terminal domain dimer and two at each of the N-terminal domains. Mutational analysis indicates that the C-terminal domain shares conserved active site architecture and DNA binding elements with the tetrameric restriction enzyme NgoMIV. Data provided here suggest possible evolutionary relationships between different subfamilies of restriction enzymes.     

The atypical chlorophyll a/b/c light-harvesting complex of Mantoniella squamata: molecular cloning and sequence analysis

cDNA species encoding precursor polypeptides of the chlorophyll a/b/c light-harvesting complex (LHC) of Mantoniella squamata were cloned and sequenced. The precursor polypeptides have molecular weights of 24.2 kDa and are related to the major chlorophyll a/b polypeptides of higher plants. Southern analysis showed that their genes belong to the nuclear encoded Lhc multigene family; the investigated genes most probably do not contain introns. The chlorophyll a/b/c polypeptides contain two highly conserved regions common to all LHC polypeptides and three hydrophobic α-helices, which span the thylakoid membrane. The first membrane-spanning helix, however, is not detected by predictive methods: its atypical hydrophilic domains may bind the chlorophyll c molecules within the hydrophobic membrane environment. Homology to LHC 11 of higher plants and green algae is specifically evident in the C-terminal region comprising helix III and the preceding stroma-exposed domain. The N-terminal region of 29 amino acids resembles the structure of a transit sequence, which shows only minor similarities to those of LHC II sequences. Strikingly, the mature light-harvesting polypeptides of M. squamata lack an N-terminal domain of 30 amino acids, which, in higher plants, contains the phosphorylation site of LHC 11 and simultaneously mediates membrane stacking. Therefore, the chlorophyll a/b/c polypeptides of M. squamata do not exhibit any light-dependent preference for photosystem I or 11. The lack of this domain also indicates that the attractive forces between stacked thylakoids are weak.     

Characterization of glutathione S-transferases from Sus scrofa,Cydia pomonella and Triticum aestivum: Their responses to cantharidin

Glutathione S-transferases (GSTs) play a key role in detoxification of xenobiotics in organisms. However, their other functions, especially response to the natural toxin cantharidin produced by beetles in the Meloidae and Oedemeridae families, are less known. We obtained GST cDNAs from three sources: Cydia pomonella (CpGSTd1), Sus scrofa (SsGSTα1), and Triticum aestivum (TaGSTf3). The predicted molecular mass is 24.19, 25.28 and 24.49 kDa, respectively. These proteins contain typical N-terminal and C-terminal domains. Recombinant GSTs were heterologously expressed in Escherichia coli as soluble fusion proteins. Their optimal activities are exhibited at pH 7.0–7.5 at 30 °C. Activity of CpGSTd1 is strongly inhibited by cantharidin and cantharidic acid, but is only slightly suppressed by the demethylated analog of cantharidin and cantharidic acid. Enzymatic assays revealed that cantharidin has no effect on SsGSTα1 activity, while it significantly stimulates TaGSTf3 activity, with an EC₅₀ value of 0.3852 mM. Activities of these proteins are potently inhibited by the known GST competitive inhibitor: S-hexylglutathione (GTX). Our results suggest that these GSTs from different sources share similar structural and biochemical characteristics. Our results also suggest that CpGSTd1 might act as a binding protein with cantharidin and its analogs.