Glutathione Binding Site Research Articles

Glutathione Non-Covalent Binding Sites on Hemoglobin and Major Glutathionylation Target betaCys93 Are Conservative among Both Hypoxia-Sensitive and Hypoxia-Tolerant Mammal Species.

Intracellular tripeptide glutathione is an important agent of cell survival under hypoxia. Glutathione covalently binds to SH groups of hemoglobin cysteine residues, protecting them from irreversible oxidation, and changes its affinity to oxygen. Reduced glutathione (GSH) can also form a noncovalent complex with hemoglobin. Previously, we showed that hemoglobin tetramer has four noncovalent binding sites of glutathione GSH molecules inside, two of which are released during hemoglobin transition to deoxy form. In this study, we characterized the conserved cysteine residues and residues of noncovalent glutathione binding sites in the sequences of a number of hypoxia-tolerant and hypoxia-sensitive mammals. The solvent accessibility of all HbA and HbB residues in oxy and deoxy forms was analyzed. The alpha subunit of all species considered was shown to have no conserved cysteines, whereas the beta subunit contains Cys93 residue, which is conserved across species and whose glutathionylation changes the affinity of hemoglobin for oxygen 5-6-fold. It was found that the key residues of noncovalent glutathione binding sites in both alpha and beta subunits are absolutely conserved in all species considered, suggesting a common mechanism of hemoglobin redox regulation for both hypoxia-sensitive and hypoxia-tolerant mammals.

Development and application of fragment-based de novo inhibitor design approaches against Plasmodium falciparum GST.

Modulation of disease progression is frequently started by identifying biochemical pathway catalyzed by biomolecule that is prone to inhibition by small molecular weight ligands. Such ligands (leads) can be obtained from natural resources or synthetic libraries. However, de novo design based on fragments assembly and optimization is showing increasing success. Plasmodium falciparum parasite depends on glutathione-S-transferase (PfGST) in buffering oxidative heme as an approach to resist some antimalarials. Therefore, PfGST is considered an attractive target for drug development. In this research, fragment-based approaches were used to design molecules that can fit to glutathione (GSH) binding site (G-site) of PfGST. The involved approaches build molecules from fragments that are either isosteric to GSH sub-moieties (ligand-based) or successfully docked to GSH binding sub-pockets (structure-based). Compared to reference GST inhibitor of S-hexyl GSH, ligands with improved rigidity, synthetic accessibility, and affinity to receptor were successfully designed. The method involves joining fragments to create ligands. The ligands were thenexplored using molecular docking, Cartesian coordinate's optimization, and simplified free energy determination as well as MD simulation and MMPBSA calculations. Several tools were used which includeOPENEYE toolkit, Open Babel, Autodock Vina, Gromacs, and SwissParam server, and molecular mechanics force field of MMFF94 for optimization and CHARMM27 for MD simulation. In addition,in-house scripts written in Matlab were used to control fragments connectionand automation of the tools.

Molecular dynamics simulations and bioinformatics’ analysis of deleterious missense single nucleotide polymorphisms in Glyoxalase-1 gene

Glyoxalase-1 (Glo-1) is a key member of the Glyoxalase system, the primary line of defense against dicarbonyl stress which, in tandem, with reduced levels of expression or activity of Glyoxalase-1 enzyme, has been implicated in various human diseases like type 2 diabetes mellitus (T2DM) and its vascular complications. The association of Glo-1 single nucleotide polymorphisms with genetic susceptibility to T2DM and its vascular complications is yet to be explored. Therefore, in this study, we have employed a computational approach to identify the most damaging missense or nonsynonymous SNPs (nsSNPs) in Glo-1 gene. Initially, we characterized missense SNPs that are damaging to the structural and functional integrity of Glo-1 using various bioinformatic tools. These tools included SIFT, PolyPhen-2, SNAP, PANTHER, PROVEAN, PhD-SNP, SNPs&GO, I-Mutant, MUpro and MutPred2. One of these missense SNPs (rs1038747749; corresponding to amino acid change Arginine to Glutamine at position 38) was found to be highly conserved in evolution and is an important part of the enzyme’s active site, glutathione binding site, as well as the dimeric interface based on the results obtained from ConSurf and NCBI Conserved Domain Search tools. Project HOPE reported that this mutation replaces a positively charged polar amino acid (Arginine) with a small, neutrally charged amino acid (Glutamine). Comparative modelling of wildtype and mutant (R38Q) Glo-1 proteins was performed in the run up to molecular dynamics simulation analysis which showed that rs1038747749 adversely impacts Glo-1 protein’s stability, rigidity, compactness, hydrogen bonds/interactions as demonstrated by the results of various parameters computed during the analysis.Communicated by Ramaswamy H. Sarma

Drugging the Undruggable Trypanosoma brucei Monothiol Glutaredoxin 1.

Trypanosoma brucei is a species of kinetoplastid causing sleeping sickness in humans and nagana in cows and horses. One of the peculiarities of this species of parasites is represented by their redox metabolism. One of the proteins involved in this redox machinery is the monothiol glutaredoxin 1 (1CGrx1) which is characterized by a unique disordered N-terminal extension exclusively conserved in trypanosomatids and other organisms. This region modulates the binding profile of the glutathione/trypanothione binding site, one of the functional regions of 1CGrx1. No endogenous ligands are known to bind this protein which does not present well-shaped binding sites, making it target particularly challenging to target. With the aim of targeting this peculiar system, we carried out two different screenings: (i) a fragment-based lead discovery campaign directed to the N-terminal as well as to the canonical binding site of 1CGrx1; (ii) a structure-based virtual screening directed to the 1CGrx1 canonical binding site. Here we report a small molecule that binds at the glutathione binding site in which the binding mode of the molecule was deeply investigated by Nuclear Magnetic Resonance (NMR). This compound represents an important step in the attempt to develop a novel strategy to interfere with the peculiar Trypanosoma Brucei redox system, making it possible to shed light on the perturbation of this biochemical machinery and eventually to novel therapeutic possibilities.

A conserved glutathione binding site in poliovirus is a target for antivirals and vaccine stabilisation

Strategies to prevent the recurrence of poliovirus (PV) after eradication may utilise non-infectious, recombinant virus-like particle (VLP) vaccines. Despite clear advantages over inactivated or attenuated virus vaccines, instability of VLPs can compromise their immunogenicity. Glutathione (GSH), an important cellular reducing agent, is a crucial co-factor for the morphogenesis of enteroviruses, including PV. We report cryo-EM structures of GSH bound to PV serotype 3 VLPs showing that it can enhance particle stability. GSH binds the positively charged pocket at the interprotomer interface shown recently to bind GSH in enterovirus F3 and putative antiviral benzene sulphonamide compounds in other enteroviruses. We show, using high-resolution cryo-EM, the binding of a benzene sulphonamide compound with a PV serotype 2 VLP, consistent with antiviral activity through over-stabilizing the interprotomer pocket, preventing the capsid rearrangements necessary for viral infection. Collectively, these results suggest GSH or an analogous tight-binding antiviral offers the potential for stabilizing VLP vaccines.

Antifilarial efficacy of andrographolide: Ex vivo studies on bovine filarial parasite Setaria cervi

Lymphatic filariasis caused by filarial nematode is an important disease leading to considerable morbidity throughout tropical countries. Even after specific elimination programs, the disease continue to spread in endemic countries. Thus newer therapeutic interventions are urgently needed to control the spread. In the present study, we have seen the effect of andrographolide (andro), a diterpenoid lactone from the leaves of Andrographis paniculata on filarial parasite Setaria cervi. There was time and concentration dependent decrease in motility and viability leading to death of parasite after 6 h of the exposure of andro. Andro showed potential antifilarial activity with an IC50 value of 24.80 μM assessed through MTT assay. There was concentration dependent decrease in the antioxidant enzymes activity and increase in proapoptotic markers after 5 h exposure of andro. Further, molecular docking analysis revealed that andro binds with filarial glutathione-S-transferase at glutathione (GSH) binding site and inhibiting enzyme activity competitively. Andro induced oxidative stress mediated apoptosis in parasites as evidenced by increase in the intracellular reactive oxygen species (ROS) and apoptotic markers.Therefore this study suggested that andro could be further explored as a new antifilarial drug.

Biochemical characterization and peptide mass fingerprinting of two glutathione transferases from Biomphalaria alexandrina snails (Gastropoda: Planorbidae)

BackgroundThe freshwater snails Biomphalaria alexandrina (Gastropoda: Planorbidae) has public health importance of being an intermediate host of Schistosoma mansoni, the parasite species that causes intestinal schistosomiasis in humans. Glutathione transferases (GSTs) play an important role in detoxification of a broad range of compounds including secondary metabolites and exogenous compounds. Studying GSTs in snails may clarify their role in detoxification of molluscicides. ResultsTwo glutathione transferases (BaGST2 and BaGST3) were purified and characterized from B. alexandrina snails. BaGST2 and BaGST3 were electrophoretically homogeneous preparations with subunit molecular weight of 23.6 kDa and molecular weight of 45 kDa. Isoelectric focusing of BaGST2 revealed the presence of two components at pI 4.47 and 4.67, while BaGST3 showed one band at pI 4.17. The specific activity of BaGST2 and BaGST3 toward 1-chloro-2,4-dinitrobenzene (CDNB) was 19.0 and 45.2 μmol/min/mg protein following 146- and 346-fold purification, respectively. The catalytic pH optima, km values, and the activation energies for BaGST2 and BaGST3 were determined. BaGST2 and BaGST3 were significantly inhibited by hematin and Cibacron Blue and to a less extent by bromosulfophthalein, S-butyl-GSH, S-hexyl-GSH, and S-P-bromobenzyl-GSH. BaGST2 and BaGST3 showed high activity against ethacrynic acid as substrate, and they also exhibited peroxidase activity on cumene hydroperoxide. The two enzymes showed identical patterns of lysine-C digestion after high-performance liquid chromatography. The amino acid sequences of three peptide fragments and peptide mass fingerprinting of fourteen peptides were used to predict the primary structure of BaGST2. A polypeptide of 206 amino acids (with 7 gaps, 3 of which could not identified) was predicted for BaGST2. The theoretical subunit molecular weight of BaGST2 is 22.6 kDa, with pI of 8.58. BaGST2 has 65% sequence identity and 78% positive with Biomphalaria glabrata GST7. The overall structure of BaGST2 at the N-terminal domain is identical to the canonical GST N-terminal domain, having the typical thioredoxin-like fold with a βαβ-α-ββα motif, whereas the C-terminal domain is made from 6 α-helices. A conservative GST-N-domain includes glutathione binding sites Y11, L17, Q53, M54, Q65, and S66, while a variable GST-C domain contains electrophilic substrate binding site H99, R102, A103, F106, K107, L161, and Y167. Phylogenetic tree showed that BaGST2 was clustered in the sigma group with GSTs sigma class from invertebrates and vertebrates. ConclusionsWe have purified and characterized two GSTs from B. alexandrina snails. Our study broadens the biochemical information on freshwater snail GSTs by demonstrating the role of BaGSTs in defense mechanisms against structurally different electrophilic compounds. BaGST2 and BaGST3 have Se-independent peroxidase activity, which indicates their role in cellular antioxidant defense by reducing organic hydroperoxides in B. alexandrina. A polypeptide chain of 206 amino acids was predicted. The primary structure of BaGST2 showed 65% sequence identity with Biomphalaria glabrata GST7. Sequence analysis indicates that BaGST2 is a GST-N-sigma-like with a thioredoxin-like superfamily. Phylogenetic tree confirms that BaGST2 belongs to the sigma class of GSTs superfamily.

Glutathione binding to the plant AtAtm3 transporter and implications for the conformational coupling of ABC transporters.

The ATP binding cassette (ABC) transporter of mitochondria (Atm) from Arabidopsis thaliana (AtAtm3) has been implicated in the maturation of cytosolic iron-sulfur proteins and heavy metal detoxification, plausibly by exporting glutathione derivatives. Using single-particle cryo-electron microscopy, we have determined four structures of AtAtm3 in three different conformational states: two inward-facing conformations (with and without bound oxidized glutathione [GSSG]), together with closed and outward-facing states stabilized by MgADP-VO4. These structures not only provide a structural framework for defining the alternating access transport cycle, but also reveal the paucity of cysteine residues in the glutathione binding site that could potentially form inhibitory mixed disulfides with GSSG. Despite extensive efforts, we were unable to prepare the ternary complex of AtAtm3 containing both GSSG and MgATP. A survey of structurally characterized type IV ABC transporters that includes AtAtm3 establishes that while nucleotides are found associated with all conformational states, they are effectively required to stabilize occluded, closed, and outward-facing conformations. In contrast, transport substrates have only been observed associated with inward-facing conformations. The absence of structures with dimerized nucleotide binding domains containing both nucleotide and transport substrate suggests that this form of the ternary complex exists only transiently during the transport cycle.

Proposed mechanism for monomethylarsonate reductase activity of human omega-class glutathione transferase GSTO1-1

Contamination of drinking water with toxic inorganic arsenic is a major public health issue. The mechanisms of enzymes and transporters in arsenic elimination are therefore of interest. The human omega-class glutathione transferases have been previously shown to possess monomethylarsonate (V) reductase activity. To further understanding of this activity, molecular dynamics of human GSTO1-1 bound to glutathione with a monomethylarsonate isostere were simulated to reveal putative monomethylarsonate binding sites on the enzyme. The major binding site is in the active site, adjacent to the glutathione binding site. Based on this and previously reported biochemical data, a reaction mechanism for this enzyme is proposed. Further insights were gained from comparison of the human omega-class GSTs to homologs from a range of animals.

Reactive oxygen species and senescence modulatory effects of rice bran extract on 4T1 and NIH-3T3 cells co-treatment with doxorubicin

Objective: To determine the effect of rice bran extract (RBE) in combination with doxorubicin on 4T1 triple-negative breast cancer cells and NIH-3T3 cells. Methods: RBE was obtained by maceration with n-hexane. The phytochemical profile of RBE was observed using highperformance liquid chromatography. Cytotoxic effect of RBE was evaluated through MTT assay. In addition, flow cytometry was used for cell cycle and apoptosis analysis. Cellular senescence was observed using SA-β-Gal assay and intracellular reactive oxygen species (ROS) levels were evaluated using DCFDA staining. The pro-oxidant property of RBE was also evaluated through 1-chloro- 2,4-dinitrobenzene spectrophotometry and molecular docking. Results: RBE was obtained with a yield of 18.42% w/w and contained tocotrienols as the major compound. RBE exerted no cytotoxic effect on 4T1 and NIH-3T3 cells. However, RBE in combination with doxorubicin decreased 4T1 cell viability synergistically (combination index<0.9) and induced apoptosis and senescence on 4T1 cells. RBE significantly decreased senescence in doxorubicin-treated NIH-3T3 cells. Additionally, RBE did not increase ROS levels in doxorubicin-treated 4T1 cells. Meanwhile, the combination of RBE and doxorubicin reduced ROS levels in NIH-3T3 cells. RBE significantly reduced glutathione-S-transferase activity and alpha-tocotrienol interacted with glutathione-S- transferase in the glutathione binding site. Conclusions: Rice bran may be used as a co-chemotherapeutic agent to improve the therapeutic effectiveness of doxorubicin while protecting against the cellular senescence effects of doxorubicin on healthy cells.

Mutational Analysis of the Binding of Alternative Substrates and Inhibitors to the Active Site of Human Glutathione Transferase P1–1

Glutathione transferases (GSTs) are enzymes that play a critical role in cellular detoxication by catalyzing the nucleophilic attack of glutathione on the electrophilic center of a number of xenobiotic compounds, including many therapeutic drugs. Mutations of amino acid residues in the glutathione-binding site of human glutathione transferase P1–1, namely W39C, K45A, Q52A, Q52K, and Q52E, have been engineered. The recombinant mutant proteins were expressed in Escherichia coli, but only mutants K45A, Q52A, and Q52K showed measurable activity. Steady-state kinetics comparing glutathione with the alternative thiol substrate γ-glutamylcysteine demonstrated the importance of the glycine residue in glutathione for high catalytic efficiency. Inhibition experiments with a set of glutathione analogs structurally related to the therapeutic drugs Telintra and Telcyta enabled determination of binding energies that were contributed by different substituents. The effects of substituting amino acid side chains in the glutathione-binding site of the enzyme on binding the glutathione derivatives and catalysis were evaluated.

The Interaction of the Microtubule Targeting Anticancer Drug Colchicine with Human Glutathione Transferases.

Glutathione transferases (GSTs) are a family of Phase II detoxification enzymes that have been shown to be involved in the development of multi-drug resistance (MDR) mechanism toward chemotherapeutic agents. GST inhibitors have, therefore, emerged as promising chemosensitizers to manage and reverse MDR. Colchicine (COL) is a classical antimitotic, tubulin-binding agent (TBA) which is being explored as anticancer drug. In the present work, the interaction of COL and its derivative 2,3-didemethylcolchicine (2,3-DDCOL) with human glutathione transferases (hGSTA1-1, hGSTP1-1, hGSTM1-1) was investigated by inhibition analysis, molecular modelling and molecular dynamics simulations. The results showed that both compounds bind reversibly to human GSTs and behave as potent inhibitors. hGSTA1-1 was the most sensitive enzyme to inhibition by COL with IC50 22 μΜ. Molecular modelling predicted that COL overlaps with both the hydrophobic (H-site) and glutathione binding site (G-site) and polar interactions appear to be the driving force for its positioning and recognition at the binding site. The interaction of COL with other members of GST family (hGSTA2-2, hGSTM3-3, hGSTM3-2) was also investigated with similar results. The results of the present study might be useful in future drug design and development efforts towards human GSTs.

A structural framework for unidirectional transport by a bacterial ABC exporter

The ATP-binding cassette (ABC) transporter of mitochondria (Atm1) mediates iron homeostasis in eukaryotes, while the prokaryotic homolog from Novosphingobium aromaticivorans (NaAtm1) can export glutathione derivatives and confer protection against heavy-metal toxicity. To establish the structural framework underlying the NaAtm1 transport mechanism, we determined eight structures by X-ray crystallography and single-particle cryo-electron microscopy in distinct conformational states, stabilized by individual disulfide crosslinks and nucleotides. As NaAtm1 progresses through the transport cycle, conformational changes in transmembrane helix 6 (TM6) alter the glutathione-binding site and the associated substrate-binding cavity. Significantly, kinking of TM6 in the post-ATP hydrolysis state stabilized by MgADPVO4 eliminates this cavity, precluding uptake of glutathione derivatives. The presence of this cavity during the transition from the inward-facing to outward-facing conformational states, and its absence in the reverse direction, thereby provide an elegant and conceptually simple mechanism for enforcing the export directionality of transport by NaAtm1. One of the disulfide crosslinked NaAtm1 variants characterized in this work retains significant glutathione transport activity, suggesting that ATP hydrolysis and substrate transport by Atm1 may involve a limited set of conformational states with minimal separation of the nucleotide-binding domains in the inward-facing conformation.

Biochemical Functions of Glutathione S-Transferase Family of Salix babylonica.

Glutathione S-transferases (GSTs) are ubiquitous enzymes that are encoded by a large gene family, and they contribute to the detoxification of endogenous or xenobiotic compounds and oxidative stress metabolism in plants. Although the GSTs gene family has been reported in many land plants, our knowledge of the evolution and function of the willow GSTs is still limited. In this study, 22 full-length GST genes were cloned from Salix babylonica and divided into three classes based on the conserved domain analysis, phylogenetic tree and gene structure: tau, phi and DHAR. The tissue-specific expression patterns were substantially different among the tau and phi GSTs. The Salix GST proteins showed functional divergences in the substrate specificities, substrate activities and kinetic characteristics. The site-directed mutagenesis studies revealed that a single amino acid mutation (Ile/Val53→Thr53) resulted in the lowest activity of SbGSTU7 among the Salix GSTs. These results suggest that non-synonymous substitution of an amino acid at the putative glutathione-binding site may play an important role in the divergence of enzymatic functions of Salix GST family.

Expression of Heterologous OsDHAR Gene Improves Glutathione (GSH)-Dependent Antioxidant System and Maintenance of Cellular Redox Status in Synechococcus elongatus PCC 7942.

An excess of reactive oxygen species (ROS) can cause severe oxidative damage to cellular components in photosynthetic cells. Antioxidant systems, such as the glutathione (GSH) pools, regulate redox status in cells to guard against such damage. Dehydroascorbate reductase (DHAR, EC 1.8.5.1) catalyzes the glutathione-dependent reduction of oxidized ascorbate (dehydroascorbate) and contains a redox active site and glutathione binding-site. The DHAR gene is important in biological and abiotic stress responses involving reduction of the oxidative damage caused by ROS. In this study, transgenic Synechococcus elongatus PCC 7942 (TA) was constructed by cloning the Oryza sativa L. japonica DHAR (OsDHAR) gene controlled by an isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible promoter (Ptrc) into the cyanobacterium to study the functional activities of OsDHAR under oxidative stress caused by hydrogen peroxide exposure. OsDHAR expression increased the growth of S. elongatus PCC 7942 under oxidative stress by reducing the levels of hydroperoxides and malondialdehyde (MDA) and mitigating the loss of chlorophyll. DHAR and glutathione S-transferase activity were higher than in the wild-type S. elongatus PCC 7942 (WT). Additionally, overexpression of OsDHAR in S. elongatus PCC 7942 greatly increased the glutathione (GSH)/glutathione disulfide (GSSG) ratio in the presence or absence of hydrogen peroxide. These results strongly suggest that DHAR attenuates deleterious oxidative effects via the glutathione (GSH)-dependent antioxidant system in cyanobacterial cells. The expression of heterologous OsDHAR in S. elongatus PCC 7942 protected cells from oxidative damage through a GSH-dependent antioxidant system via GSH-dependent reactions at the redox active site and GSH binding site residues during oxidative stress.

Two sigma and two mu class genes of glutathione S-transferase in the waterflea Daphnia pulex: Molecular characterization and transcriptional response to nanoplastic exposure

Two isoforms of Glutathione S-Transferase (GST) genes, belonging to mu (Dp-GSTm1 and Dp-GSTm2) and sigma (Dp-GSTs1 and Dp-GSTs2) classes, were cloned and characterised in the freshwater Daphnia pulex. No signal peptide was found in any of the four GST proteins, indicating that they were cytosolic GST. A highly conserved glutathione (GSH) binding site (G-site) occurred in the N-terminal sequence, and a substrate binding site (H-site), interacting non-specifically with the second hydrophobic substrate, was present in the C-terminal. A Tyr residue, for the stabilization of GSH, was found to be conserved in the analysed sequences. The secondary and tertiary structures indicated that these genes possess the typical cytosolic GST structure, including a conserved N-terminal domain with a βαβαββα motif. The μ loop (NVGPAPDYDR and NFIGAEWDR in Dp-GSTm1 and Dp-GSTm2, respectively) was identified between the βαβ (β1α1β2) and αββα motifs (α2β3β4α3) in the N-terminal domain. The expressions of Dp-GSTs1, Dp-GSTs2, and Dp-GSTm1 were higher in other age groups compared to the newly-born neonates (1 d); however, the expression of Dp-GSTm2 first increased and then decreased with age. Gene expression was significantly reduced by high concentration (1 and 2 mg/L) of 75 nm polystyrene nanoplastic. However, nanoplastic exposure at the predicted environmental concentration (1 μg/L) had a low effect. Exposure of mothers to nanoplastic (1 μg/L) elevated the Dp-GSTs2 level in their neonates. These results improve our understanding on the response of different types of Daphnid GST to environmental contaminants, especially nanoplastic.

Phylogenetic distribution and structural analyses of cyanobacterial glutaredoxins (Grxs)

Glutaredoxins (Grxs), the oxidoreductase proteins, are involved in several cellular processes, including maintenance of cellular redox potential and iron-sulfur homeostasis. The analysis of 503 amino acid sequences from 167 cyanobacterial species led to the identification of four classes of cyanobacterial Grxs, i.e., class I, II, V, and VI Grxs. Class III and IV Grxs were absent in cyanobacteria. Class I and II Grxs are single module oxidoreductase while class V and VI Grxs are multimodular proteins having additional modules at their C-terminal and N-terminal end, respectively. Furthermore, class VI Grxs were exclusively present in marine cyanobacteria. We also report the identification of class VI Grxs with two novel active site motif compositions. Detailed phylogenetic analysis of all four classes of Grxs revealed the presence of several subgroups within each class of Grx having variable dithiol and/or monothiol catalytic active site motif and putative glutathione binding sites. However, class II Grxs possess CGFS-type highly conserved monothiol catalytic active site motif. Sequence analysis confirmed the highly diverse nature of Grx proteins in terms of their amino acid composition; though, sequence diversity does not affect the overall 3D structure of cyanobacterial Grxs. The active site residues and putative GSH binding residues are uncharged amino acids which are present on the surface of the protein. Additionally, the presence of hydrophilic residues at the surface of Grxs confirms their solubility. Protein-ligand interaction analysis identified novel glutathione binding sites on Grxs. Regulation of Grxs encoding genes expression by light quality and quantity as well as salinity suggests their role in determining the fitness of organisms under abiotic factors.

Immune and xenobiotic responses of glutathione S-Transferase theta (GST-θ) from marine invertebrate disk abalone (Haliotis discus discus): With molecular characterization and functional analysis

Representing a multifunctional complex group of proteins, glutathione S- transferases (GSTs) play a major role in the phase II detoxification process in a wide range of organisms. This study focused on the potential detoxification ability of disk abalone (Haliotis discus discus) GST theta (AbGST-θ) under different stress conditions with special reference to post immune challenges. Characterization of AbGST-θ revealed with 226 amino acids, 26.6 kDa of predicted molecular mass and 8.9 of theoretical isoelectric point. As illustrated in the multiple sequence alignment, eight glutathione binding sites (G-sites) and ten substrate binding sites (H-sites) were identified in well-distinct N-terminal and C-terminal domains of AbGST-θ, respectively. AbGST-θ exhibited its highest sequence identity with Mizuhopecten yessoensis (59.1%) and the phylogenetic tree clearly positioned AbGST-θ with pre-defined GST-θ molluscan homologues. The AbGST-θ was highly expressed in the digestive tract of un-challenged abalones. Upon administering the challenge experiment, AbGST-θ showed significant modulations in their transcriptional levels depending on the time and the tissue type. The optimum temperature was 37 °C and optimum pH was 7.5 for AbGST-θ. The determined enzyme kinetic parameters of AbGST-θ showed low affinity towards 1-Chloro-2,4-dinitrobenzene (CDNB) and glutathione (GSH) as substrates. Nonetheless, with Cibacron blue IC50 (half maximal inhibitory concentration) was calculated to be 0.08 μM while observing 100% inhibition with 100 μM. Furthermore, AbGST-θ resulted in significant protection ability towards H2O2, CdCl2, and ZnCl2 in the disk diffusion assay. Collectively, this study provides evidences for the detoxification ability and the immunological host defensive capability of AbGST-θ in disk abalone.

Deciphering the number and location of active sites in the monomeric glyoxalase I of Zea mays.

Detoxification of methylglyoxal, a toxic by-product of central sugar metabolism, is a major issue for all forms of life. The glyoxalase pathway evolved to effectively convert methylglyoxal into d-lactate via a glutathione hemithioacetal intermediate. Recently, we have shown that the monomeric glyoxalase I from maize exhibits a symmetric fold with two cavities, potentially harboring two active sites, in analogy with homodimeric enzyme surrogates. Here we confirm that only one of the two cavities exhibits glyoxalase I activity and show that it adopts a tunnel-shaped structure upon substrate binding. Such conformational change gives rise to independent binding sites for glutathione and methylglyoxal in the same active site, with important implications for the molecular reaction mechanism, which has been a matter of debate for several decades. DATABASE: Structural data are available in The Protein Data Bank database under the accession numbers 6BNN, 6BNX, and 6BNZ.

Copper-induced oxidative cleavage of glutathione transferase F1-1 from Zea mays.

Study of the interaction of glutathione transferase F1-1 from Zea mays (ZmGSTF1-1) with Cu(II), in the presence of ascorbate showed that the enzyme was rapidly inactivated. The inactivation was time and Cu(II) concentration dependent. The rate of inactivation showed non-linear dependence on Cu(II) concentration, indicating that a reversible complex with the enzyme (KD 84.5 ± 6.5 μM) was formed. The inhibitors S-nitrobenzyl-glutathione or S-methyl-glutathione competes with Cu(II), suggesting the specificity of the chemical modification reaction. SDS-PAGE analysis of the inactivated enzyme showed that the enzyme is fragmented and two new bands of 13 and 11 kDa are formed. This shows that ZmGSTF1-1 was specifically cleaved at a single site, by the locally generated free radicals, through a Fenton-type reaction. Sequencing of the fragments allowed the identification of the Cu(II) binding site on ZmGSTF1-1. The three-dimensional structure of ZmGSTF1-1 reveals that the Cu(II) binding site is localized within the glutathione-binding site (G-site) and His40 and Gln53 are most likely the residues that provide the coordination sites for the Cu(II) binding. These findings were confirmed by site-directed mutagenesis. This copper-induced oxidative cleavage reaction of ZmGSTF1-1 may function as a detoxification route for Cu(II) for protecting plant cells from copper-induced deleterious effects.