Understanding nicotinamide dinucleotide cofactor and substrate specificity in class I flavoprotein disulfide oxidoreductases: crystallographic analysis of a glutathione amide reductase

Glutathione reductase (GR) plays a vital role in maintaining the antioxidant levels of the cytoplasm by catalyzing the reduction of glutathione disulfide to reduced glutathione, thereby using NADPH and flavin adenine dinucleotide as cofactors. Chromatiaceae have evolved an unusual homolog that prefers both a modified substrate (glutathione amide disulfide [GASSAG]) and a different cofactor (NADH). Herein, we present the crystal structure of the Chromatium gracile glutathione amide reductase (GAR) both alone and in complex with NAD⁺. An altered charge distribution in the GASSAG binding pocket explains the difference in substrate specificity. The NADH binding pocket of GAR differs from that of wild-type GR as well as that of a low active GR that was engineered to mimic NADH binding. Based on the GAR structure, we propose two attractive rationales for producing an efficient GR enzyme with NADH specificity.     

Structural basis for the different activities of yeast Grx1 and Grx2

Yeast glutaredoxins Grx1 and Grx2 catalyze the reduction of both inter- and intra-molecular disulfide bonds using glutathione (GSH) as the electron donor. Although sharing the same dithiolic CPYC active site and a sequence identity of 64%, they have been proved to play different roles during oxidative stress and to possess different glutathione-disulfide reductase activities. To address the structural basis of these differences, we solved the crystal structures of Grx2 in oxidized and reduced forms, at 2.10 Å and 1.50 Å, respectively. With the Grx1 structures we previously reported, comparative structural analyses revealed that Grx1 and Grx2 share a similar GSH binding site, except for a single residue substitution from Asp89 in Grx1 to Ser123 in Grx2. Site-directed mutagenesis in combination with activity assays further proved this single residue variation is critical for the different activities of yeast Grx1 and Grx2.     

Regulation of antioxidant metabolism by translation initiation factor 2alpha

Oxidative stress and highly specific decreases in glutathione (GSH) are associated with nerve cell death in Parkinson's disease. Using an experimental nerve cell model for oxidative stress and an expression cloning strategy, a gene involved in oxidative stress-induced programmed cell death was identified which both mediates the cell death program and regulates GSH levels. Two stress-resistant clones were isolated which contain antisense gene fragments of the translation initiation factor (eIF)2alpha and express a low amount of eIF2alpha. Sensitivity is restored when the clones are transfected with full-length eIF2alpha; transfection of wild-type cells with the truncated eIF2alpha gene confers resistance. The phosphorylation of eIF2alpha also results in resistance to oxidative stress. In wild-type cells, oxidative stress results in rapid GSH depletion, a large increase in peroxide levels, and an influx of Ca(2+). In contrast, the resistant clones maintain high GSH levels and show no elevation in peroxides or Ca(2+) when stressed, and the GSH synthetic enzyme gamma-glutamyl cysteine synthetase (gammaGCS) is elevated. The change in gammaGCS is regulated by a translational mechanism. Therefore, eIF2alpha is a critical regulatory factor in the response of nerve cells to oxidative stress and in the control of the major intracellular antioxidant, GSH, and may play a central role in the many neurodegenerative diseases associated with oxidative stress.     

Structural basis for detoxification and oxidative stress protection in membranes

Synthesis of mediators of fever, pain and inflammation as well as protection against reactive molecules and oxidative stress is a hallmark of the MAPEG superfamily (membrane associated proteins in eicosanoid and glutathione metabolism). The structure of a MAPEG member, rat microsomal glutathione transferase 1, at 3.2 A resolution, solved here in complex with glutathione by electron crystallography, defines the active site location and a cytosolic domain involved in enzyme activation. The glutathione binding site is found to be different from that of the canonical soluble glutathione transferases. The architecture of the homotrimer supports a catalytic mechanism involving subunit interactions and reveals both cytosolic and membraneous substrate entry sites, providing a rationale for the membrane location of the enzyme.     

Plasmodium falciparum antioxidant protein as a model enzyme for a special class of glutaredoxin/glutathione-dependent peroxiredoxins

Background

Peroxiredoxins are important heterogeneous thiol-dependent hydroperoxidases with a variety of isoforms and enzymatic mechanisms. A special subclass of glutaredoxin/glutathione-dependent peroxiredoxins has been discovered in bacteria and eukaryotes during the last decade, but the exact enzymatic mechanisms of these enzymes remain to be unraveled.

Methods

We performed a comprehensive analysis of the enzyme kinetics and redox states of one of these glutaredoxin/glutathione-dependent peroxiredoxins, the antioxidant protein from the malaria parasite Plasmodium falciparum, using steady-state kinetic measurements, site-directed mutagenesis, redox mobility shift assays, gel filtration, and mass spectrometry.

Results

P. falciparum antioxidant protein requires not only glutaredoxin but also glutathione as a true substrate for the reduction of hydroperoxides. One peroxiredoxin cysteine residue and one glutaredoxin cysteine residue are sufficient for catalysis, however, additional cysteine residues of both proteins result in alternative redox states and conformations in vitro with implications for redox regulation. Our data furthermore point to a glutathione-dependent peroxiredoxin activation and a negative subunit cooperativity.

Conclusions

The investigated glutaredoxin/glutathione/peroxiredoxin system provides numerous new insights into the mechanism and redox regulation of peroxiredoxins.

General significance

As a member of the special subclass of glutaredoxin/glutathione-dependent peroxiredoxins, the P. falciparum antioxidant protein could become a reference protein for peroxiredoxin catalysis and regulation.     

Properties and functions of glutathione reductase in plants

The assay and in vitro characterization of glutathione reductase (EC 1.6.4.2) is discussed. In vivo the H₂O₂-scavenging system in chloroplasts is the best documented role of reduced glutathione and glutathione reductase in plants. Similarly, redaction of H₂O₂, outside of the chloroplasts, requires glutathione and glutathione reductase; but the pathway, in terms of intermediates, is controversial. The notion that biological stress frequently causes cellular oxidation has lead to the suggestion that glutathione and glutathione reductase may play a role in stress resistance or tolerance mechanisms. The changes in glutathione reductase levels in response to low temperature, oxidative stress and drought are discussed.     

The evolution of catalytic efficiency and substrate promiscuity in human theta class 1-1 glutathione transferase

Theta class glutathione transferases (GST) from various species exhibit markedly different catalytic activities in conjugating the tripeptide glutathione (GSH) to a variety of electrophilic substrates. For example, the human theta 1-1 enzyme (hGSTT1-1) is 440-fold less efficient than the rat theta 2-2 enzyme (rGSTT2-2) with the fluorogenic substrate 7-amino-4-chloromethyl coumarin (CMAC). Large libraries of hGSTT1-1 constructed by error-prone PCR, DNA shuffling, or saturation mutagenesis were screened for improved catalytic activity towards CMAC in a quantitative fashion using flow cytometry. An iterative directed evolution approach employing random mutagenesis in conjunction with homologous recombination gave rise to enzymes exhibiting up to a 20,000-fold increase in k(cat)/K(M) compared to hGSTT1-1. All highly active clones encoded one or more mutations at residues 32, 176, or 234. Combinatorial saturation mutagenesis was used to evaluate the full complement of natural amino acids at these positions, and resulted in the isolation of enzymes with catalytic rates comparable to those exhibited by the fastest mutants obtained via directed evolution. The substrate selectivities of enzymes resulting from random mutagenesis, DNA shuffling, and combinatorial saturation mutagenesis were evaluated using a series of distinct electrophiles. The results revealed that promiscuous substrate activities arose in a stochastic manner, as they did not correlate with catalytic efficiency towards the CMAC selection substrate. In contrast, chimeric enzymes previously constructed by homology-independent recombination of hGSTT-1 and rGSTT2-2 exhibited very different substrate promiscuity profiles, and showed a more defined relationship between evolved and promiscuous activities.     

In vitro Interactions of Thallium with Components of the Glutathione-dependent Antioxidant Defence System

We investigated the hypothesis that thallium (Tl) interactions with the glutathione-dependent antioxidant defence system could contribute to the oxidative stress associated with Tl toxicity. Working in vitro with reduced glutathione (GSH), glutathione reductase (GR) or glutathione peroxidase (GPx) in solution, we studied the effects of Tl⁺ and Tl³⁺ (1-25 μM) on: (a) the amount of free GSH, investigating whether the metal binds to GSH and/or oxidizes it; (b) the activity of the enzyme GR, that catalyzes GSH regeneration; and (c) the enzyme GPx, that reduces hydroperoxide at expense of GSH oxidation. We found that, while Tl⁺ had no effect on GSH concentration, Tl³⁺ oxidized it. Both cations inhibited the reduction of GSSG by GR and the diaphorase activity of this enzyme. In addition, Tl³⁺per se oxidized NADPH, the cofactor of GR. The effects of Tl on GPx activity depended on the metal charge: Tl⁺ inhibited GPx when cumene hydroperoxide (CuOOH) was the substrate, while Tl³⁺-mediated GPx inhibition occurred with both substrates. The present results show that Tl interacts with all the components of GSH/GSSG antioxidant defence system. Alterations of this protective pathway could be partially responsible for the oxidative stress associated with Tl toxicity.     

Evaluation of glutathione S-transferase genetic variants affecting type 2 diabetes susceptibility: A meta-analysis

Genetic polymorphisms of glutathione S-transferases (GSTs) and type 2 diabetes mellitus (T2DM) risk have been widely studied, however, the results were somewhat conflicting. To evaluate the association of GSTs (GSTM1, GSTT1 and GSTP1) gene polymorphisms with T2DM, a meta-analysis was performed before October, 2012. ORs were pooled according to random-effects model. There were a total of 1354/1666 (n = 9) cases/controls (studies) for GSTM1, 1271/1470 (n = 8) for GSTT1, and 1205/1250 (n = 7) for GSTM1. There were significant associations between GSTM1 polymorphism, GSTT1 polymorphism and T2DM in the contrast of present genotype vs. null genotype, with pooled OR = 1.99 (95%CI = 1.46–2.71) and OR = 1.61 (95%CI = 1.19–2.17), respectively. Yet no significant association of GSTP1 polymorphism and T2DM was showed. When stratified by ethnicity, the significant associations were also existed in Asians for GSTM1 and GSTT1, but not GSTP1. No publication bias but some extent of heterogeneity was observed. Finally, the accumulated evidence proved the obvious associations of GSTM1 and GSTT1 polymorphisms with an increased risk of T2DM.     

Predissociated dimers and molten globule monomers in the equilibrium unfolding of yeast glutathione reductase

The equilibrium unfolding of dimeric yeast glutathione reductase (GR) by guanidine hydrochloride (GdnHCl) was investigated. Unfolding was monitored by a variety of techniques, including intrinsic fluorescence emission, anisotropy and iodide quenching measurements, far-ultraviolet circular dichroism and thiol reactivity measurements. At 1 M GdnHCl, one thiol group of GR became accessible to modification with 5,5′-dithiobis-(2-nitrobenzoic) acid (DTNB), whereas no changes could be detected in the spectroscopic properties (fluorescence, circular dichroism) of the protein. Between 2 and 3 M GdnHCl, two partially folded intermediate states possessing flexible tertiary structures (revealed by fluorescence data) but compact secondary structures (as indicated by circular dichroism measurements) were identified. The quaternary structure of GR in the presence of GdnHCl was also investigated by size-exclusion liquid chromatography. These results indicated the presence of an expanded predissociated dimer at 2.5 M GdnHCl and partially folded monomers at 3 M GdnHCl. Taken together, these results suggest the existence of two molten-globule-like intermediate species (one dimeric and one monomeric) in the unfolding of GR. The results are discussed in terms of the mechanism of GR folding and dimerization.     

The S. cerevisiae Yap1 and Yap2 transcription factors share a common cadmium-sensing domain

Towards elucidating the function of Yap2, which remains unclear, we have taken advantage of the C-terminal homology between Yap1 and Yap2. Swapping domains experiments show that the Yap2 C-terminal domain functionally substitutes for the homologous Yap1 domain in the response to Cd, but not to H2O2. We conclude that specificity determinants of the Cd response are encoded within both Yap1 and Yap2 C-terminus, whereas those required for H2O2 response are only present in the Yap1 C-terminus. Furthermore, our results identify FRM2 as Cd-responsive Yap2 target and indicate a possible role of this protein in regulating a metal stress response.     

α-Synuclein-mediated defense against oxidative stress via modulation of glutathione peroxidase

In this report, mutual effect of α-synuclein and GPX-1 is investigated to unveil their involvement in the PD pathogenesis in terms of cellular defense mechanism against oxidative stress. Biochemical and immunocytochemical studies showed that α-synuclein enhanced the GPX-1 activity with Kd of 17.3 nM and the enzyme in turn markedly enhanced in vitro fibrillation of α-synuclein. Transmission electron microscopy revealed the fibrillar meshwork of α-synuclein containing GPX-1 located in locally concentrated islets. The entrapped enzyme was demonstrated to be protected in a latent form and its activity was fully recovered as released from the matrix. Therefore, novel defensive roles of α-synuclein and its amyloid fibrils against oxidative stress are suggested as the GPX-1 stimulator and the active depot for the enzyme, respectively.     

Glutathione peroxidase activity,selenium, and lipid peroxide concentrations in blood from a healthy Polish population

Selenium (Se) concentrations in whole blood and plasma of 19 nonpregnant women. 14 mothers at delivery, 14 neonates, and 13 infants, aged 2–12 mo, were evaluated. The activity of glutathione peroxidase (GSH-Px) in erythrocytes and plasma and the level of lipid peroxides in plasma were also analyzed. Selenium concentrations in whole blood and plasma in mothers at delivery were significantly lower compared to nonpregnant women. Selenium concentrations in cord blood components were lower compared to mothers, but the differences were not significant. The concentration of the element decreased in the first few months of life. Glutathione peroxidase activity in erythrocytes differed only slightly in the examined groups. In plasma, however, the enzyme activity was significantly lower in pregnant compared to nonpregnant women and in neonates compared to their mothers. Lipid peroxide concentrations in plasma differed only slightly in the examined groups. The results obtained are discussed in terms of the observations of other investigators.     

The cellular redox state as a modulator in cadmium and copper responses in Arabidopsis thaliana seedlings

The cellular redox state is an important determinant of metal phytotoxicity. In this study we investigated the influence of cadmium (Cd) and copper (Cu) stress on the cellular redox balance in relation to oxidative signalling and damage in Arabidopsis thaliana. Both metals were easily taken up by the roots, but the translocation to the aboveground parts was restricted to Cd stress. In the roots, Cu directly induced an oxidative burst, whereas enzymatic ROS (reactive oxygen species) production via NADPH oxidases seems important in oxidative stress caused by Cd. Furthermore, in the roots, the glutathione metabolism plays a crucial role in controlling the gene regulation of the antioxidative defence mechanism under Cd stress. Metal-specific alterations were also noticed with regard to the microRNA regulation of CuZnSOD gene expression in both roots and leaves. The appearance of lipid peroxidation is dual: it can be an indication of oxidative damage as well as an indication of oxidative signalling as lipoxygenases are induced after metal exposure and are initial enzymes in oxylipin biosynthesis.In conclusion, the metal-induced cellular redox imbalance is strongly dependent on the chemical properties of the metal and the plant organ considered. The stress intensity determines its involvement in downstream responses in relation to oxidative damage or signalling.     

Glutathione and glutathione S-transferases in the salmonella mammalian-microsome mutagenicity test

Levels of the tripeptide glutathione (GSH) and the activity of glutathione S-transferases were investigated in S9 fractions of rats and mice and in Salmonella typhymurium tester strains TA1535, TA100, TA1538 and TA98. The S9 and Salmonella typhimurium tester strains had high levels of glutathione. Compared with S9, the activity of GSH S-transferases was lower in the bacteria. However, electrophiles such as 1-chloro-2,4-dinitrobenzene (CDNB), diethyl maleate and styrene oxide were effectively bound to bacterial GSH.

The mutagenicity of the direct mutagen CDNB was drastically lowered in presence of S9 fractions but not in presence of microsomes. A comparable decrease was obtained when microsomal supernatant, which contains GSH and GSH S-transferases, was added to the microsomes. Addition of GSH in excess completely abolished mutagenicity of CDNB. These results demonstrate that the conjugation of electrophiles with GSH mediated by the S9 fraction or the bacterial tester strains represents an important detoxication mechanism which may influence the results obtained with the Salmonella typhimurium mammalian-microsome mutagenicity test.     

Catalytic cycle of human glutathione reductase near 1 A resolution

Efficient enzyme catalysis depends on exquisite details of structure beyond those resolvable in typical medium- and high-resolution crystallographic analyses. Here we report synchrotron-based cryocrystallographic studies of natural substrate complexes of the flavoenzyme human glutathione reductase (GR) at nominal resolutions between 1.1 and 0.95 Å that reveal new aspects of its mechanism. Compression in the active site causes overlapping van der Waals radii and distortion in the nicotinamide ring of the NADPH substrate, which enhances catalysis via stereoelectronic effects. The bound NADPH and redox-active disulfide are positioned optimally on opposite sides of the flavin for a 1,2-addition across a flavin double bond. The new structures extend earlier observations to reveal that the redox-active disulfide loop in GR is an extreme case of sequential peptide bonds systematically deviating from planarity—a net deviation of 53° across five residues. But this apparent strain is not a factor in catalysis, as it is present in both oxidized and reduced structures. Intriguingly, the flavin bond lengths in oxidized GR are intermediate between those expected for oxidized and reduced flavin, but we present evidence that this may not be due to the protein environment but instead due to partial synchrotron reduction of the flavin by the synchrotron beam. Finally, of more general relevance, we present evidence that the structures of synchrotron-reduced disulfide bonds cannot generally be used as reliable models for naturally reduced disulfide bonds.     

Neuronal damage and cognitive impairment associated with hypoglycemia: An integrated view

The aim of the present review is to offer a current perspective about the consequences of hypoglycemia and its impact on the diabetic disorder due to the increasing incidence of diabetes around the world. The main consequence of insulin treatment in type 1 diabetic patients is the occurrence of repetitive periods of hypoglycemia and even episodes of severe hypoglycemia leading to coma. In the latter, selective neuronal death is observed in brain vulnerable regions both in humans and animal models, such as the cortex and the hippocampus. Cognitive damage subsequent to hypoglycemic coma has been associated with neuronal death in the hippocampus. The mechanisms implicated in selective damage are not completely understood but many factors have been identified including excitotoxicity, oxidative stress, zinc release, PARP-1 activation and mitochondrial dysfunction. Importantly, the diabetic condition aggravates neuronal damage and cognitive failure induced by hypoglycemia. In the absence of coma prolonged and severe hypoglycemia leads to increased oxidative stress and discrete neuronal death mainly in the cerebral cortex. The mechanisms responsible for cell damage in this condition are still unknown. Recurrent moderate hypoglycemia is far more common in diabetic patients than severe hypoglycemia and currently important efforts are being done in order to elucidate the relationship between cognitive deficits and recurrent hypoglycemia in diabetics. Human studies suggest impaired performance mainly in memory and attention tasks in healthy and diabetic individuals under the hypoglycemic condition. Only scarce neuronal death has been observed under moderate repetitive hypoglycemia but studies suggest that impaired hippocampal synaptic function might be one of the causes of cognitive failure. Recent studies have also implicated altered mitochondrial function and mitochondrial oxidative stress.     

Transient light-induced intracellular oxidation revealed by redox biosensor

We have implemented a ratiometric, genetically encoded redox-sensitive green fluorescent protein fused to human glutaredoxin (Grx1-roGFP2) to monitor real time intracellular glutathione redox potentials of mammalian cells. This probe enabled detection of media-dependent oxidation of the cytosol triggered by short wavelength excitation. The transient nature of light-induced oxidation was revealed by time-lapse live cell imaging when time intervals of less than 30 s were implemented. In contrast, transient ROS generation was not observed with the parental roGFP2 probe without Grx1, which exhibits slower thiol-disulfide exchange. These data demonstrate that the enhanced sensitivity of the Grx1-roGFP2 fusion protein enables the detection of short-lived ROS in living cells. The superior sensitivity of Grx1-roGFP2, however, also enhances responsiveness to environmental cues introducing a greater likelihood of false positive results during image acquisition.     

Crystal structures of a poplar thioredoxin peroxidase that exhibits the structure of glutathione peroxidases: insights into redox-driven conformational changes

Glutathione peroxidases (GPXs) are a group of enzymes that regulate the levels of reactive oxygen species in cells and tissues, and protect them against oxidative damage. Contrary to most of their counterparts in animal cells, the higher plant GPX homologues identified so far possess cysteine instead of selenocysteine in their active site. Interestingly, the plant GPXs are not dependent on glutathione but rather on thioredoxin as their in vitro electron donor. We have determined the crystal structures of the reduced and oxidized form of Populus trichocarpaxdeltoides GPX5 (PtGPX5), using a selenomethionine derivative. PtGPX5 exhibits an overall structure similar to that of the known animal GPXs. PtGPX5 crystallized in the assumed physiological dimeric form, displaying a pseudo ten-stranded beta sheet core. Comparison of both redox structures indicates that a drastic conformational change is necessary to bring the two distant cysteine residues together to form an intramolecular disulfide bond. In addition, a computer model of a complex of PtGPX5 and its in vitro recycling partner thioredoxin h1 is proposed on the basis of the crystal packing of the oxidized form enzyme. A possible role of PtGPX5 as a heavy-metal sink is also discussed.