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At least 19 recordsLinked to original sources

PubMed1992

NMR structure of oxidized Escherichia coli glutaredoxin: comparison with reduced E. coli glutaredoxin and functionally related proteins.

The determination of the NMR structure of oxidized Escherichia coli glutaredoxin in aqueous solution is described, and comparisons of this structure with that of reduced E. coli glutaredoxin and the related proteins E. coli thioredoxin and T4 glutaredoxin are presented. Based on nearly complete sequence-specific 1H-NMR assignments, 804 nuclear Overhauser enhancement distance constraints and 74 dihedral angle constraints were obtained as the input for the structure calculations, for which the distance geometry program DIANA was used followed by simulated annealing with the program X-PLOR. The molecular architecture of oxidized glutaredoxin is made up of three helices and a four-stranded beta-sheet. The three-dimensional structures of oxidized and the recently described reduced glutaredoxin are very similar. Quantitative analysis of the exchange rates of 34 slowly exchanging amide protons from corresponding series of two-dimensional [15N,1H]-correlated spectra of oxidized and reduced glutaredoxin showed close agreement, indicating almost identical hydrogen-bonding patterns. Nonetheless, differences in local dynamics involving residues near the active site and the C-terminal alpha-helix were clearly manifested. Comparison of the structure of E. coli glutaredoxin with those of T4 glutaredoxin and E. coli thioredoxin showed that all three proteins have a similar overall polypeptide fold. An area of the protein surface at the active site containing Arg 8, Cys 11, Pro 12, Tyr 13, Ile 38, Thr 58, Val 59, Pro 60, Gly 71, Tyr 72, and Thr 73 is proposed as a possible site for interaction with other proteins, in particular ribonucleotide reductase. It was found that this area corresponds to previously proposed interaction sites in T4 glutaredoxin and E. coli thioredoxin. The solvent-accessible surface area at the active site of E. coli glutaredoxin showed a general trend to increase upon reduction. Only the sulfhydryl group of Cys 11 is exposed to the solvent, whereas that of Cys 14 is buried and solvent inaccessible.

Amino Acid Sequence↗

PubMed2004

Nuclear monothiol glutaredoxins of Saccharomyces cerevisiae can function as mitochondrial glutaredoxins.

Glutaredoxins are thiol oxidoreductases that regulate protein redox state. In Saccharomyces cerevisiae, Grx1 and Grx2 are cytosolic dithiol glutaredoxins, whereas Grx3, Grx4, and Grx5 are monothiol glutaredoxins. Grx5 locates at the mitochondrial matrix and is needed for iron/sulfur cluster biogenesis. Its absence causes phenotypes such as inactivation of iron/sulfur enzymes and sensitivity to oxidative stress. Whereas Grx5 contains a single glutaredoxin domain, in Grx3 and Grx4 a thioredoxin-like domain is fused to the glutaredoxin domain. Here we have shown that Grx3 locates at the nucleus and that the thioredoxin-like domain is required for such location. We have addressed the functional divergence among glutaredoxins by targeting Grx2/3/4 molecules to the mitochondrial matrix using the Grx5 targeting sequence. The mitochondrial forms of Grx3 and Grx4 partially rescue the defects of a grx5 null mutant. On the contrary, mitochondrially targeted Grx2 does not suppress the mutant phenotype. Both the thioredoxin-like and glutaredoxin domains are needed for the mitochondrial activity of Grx3, although none of the cysteine residues at the thioredoxin-like domain is required for rescue of the grx5 phenotypes. We have concluded that dithiol glutaredoxins are functionally divergent from monothiol ones, but the latter can interchange their biological activities when compartment barriers are surpassed.

Aconitate Hydratase↗

PubMed1997

Cloning, overexpression, and characterization of glutaredoxin 2, an atypical glutaredoxin from Escherichia coli.

Glutaredoxin 2 (Grx2) from Escherichia coli catalyzes GSH-disulfide oxidoreductions via two redox-active cysteine residues, but in contrast to glutaredoxin 1 (Grx1) and glutaredoxin 3 (Grx3), is not a hydrogen donor for ribonucleotide reductase. To characterize Grx2, a chromosomal fragment containing the E. coli Grx2 gene (grxB) was cloned and sequenced. grxB (645 base pairs) is located between the rimJ and pyrC genes while an open reading frame immediately upstream grxB encodes a novel transmembrane protein of 402 amino acids potentially belonging to class II of substrate export transporters. The deduced amino acid sequence for Grx2 comprises 215 residues with a molecular mass of 24.3 kDa. There is almost no similarity between the amino acid sequence of Grx2 and Grx1 or Grx3 (both 9-kDa proteins) with the exception of the active site which is identical in all three glutaredoxins (C9PYC12 for Grx2). Only limited similarities were noted to glutathione S-transferases (Grx2 amino acids 16-72), and protein disulfide isomerases from different organisms (Grx2 amino acids 70-180). Grx2 was overexpressed and purified to homogeneity and its activity was compared with those of Grx1 and Grx3 using GSH, NADPH, and glutathione reductase in the reduction of 0.7 mM beta-hydroxyethyl disulfide. The three glutaredoxins had similar apparent Km values for GSH (2-3 mM) but Grx2 had the highest apparent kcat (554 s-1). Expression of two truncated forms of Grx2 (1-114 and 1-133) which have predicted secondary structures similar to Grx1 (betaalphabetaalphabetabetaalpha) gave rise to inclusion bodies. The mutant proteins were resolubilized and purified but lacked GSH-disulfide oxidoreductase activity. The latter should therefore require the participation of amino acid residues from the COOH-terminal half of the molecule and is probably not confined to a Grx1-like NH2-terminal subdomain. Grx2 being radically different from the presently known glutaredoxins in terms of molecular weight, amino acid sequence, catalytic activity, and lack of a consensus GSH-binding site is the first member of a novel class of glutaredoxins.

Amino Acid Sequence↗

PubMed2001

Plasmodium falciparum possesses a classical glutaredoxin and a second, glutaredoxin-like protein with a PICOT homology domain.

The genes coding for two different proteins with homologies to glutaredoxins have been identified in the genome of the malarial parasite Plasmodium falciparum. Both genes were amplified from a gametocytic cDNA and overexpressed in Escherichia coli. The smaller protein (named PfGrx-1) with 12.4 kDa in size exhibits the typical glutaredoxin active site motif "CPYC," shows glutathione-dependent glutaredoxin activity in the beta-hydroxyethyl disulfide (HEDS) assay, and reduces Trypanosoma brucei ribonucleotide reductase. Glutathione:HEDS transhydrogenase activity (approximately 60 milliunits/mg of protein) was clearly detectable in trophozoite extracts from eight different P. falciparum strains and did not differ between chloroquine-resistant and -sensitive parasites. Five different antimalarial drugs at 100 microm did not significantly influence isolated PfGrx-1 activity. In contrast, the second protein (deduced mass 19.9 kDa) with homology to glutaredoxins (31% identity to Schizosaccharomyces pombe in a 140-amino acid overlap) was not active in the HEDS assay; however, its general dithiol reducing activity was demonstrated in the insulin assay in the presence of dithiothreitol. Interestingly, the sequence contains a PICOT (for protein kinase C-interacting cousin of thioredoxin) homology domain, which might suggest regulatory functions of the protein. We named this protein PfGLP-1, for P. falciparum 1-Cys-glutaredoxin-like protein-1. In contrast to glutaredoxins, PfGLP-1 could not be reduced by glutathione. This is the first report on glutaredoxin-like proteins in the family of Plasmodia.

Amino Acid Sequence↗

PubMed1999

Reactivity of glutaredoxins 1, 2, and 3 from Escherichia coli shows that glutaredoxin 2 is the primary hydrogen donor to ArsC-catalyzed arsenate reduction.

In Escherichia coli ArsC catalyzes the reduction of arsenate to arsenite using GSH with glutaredoxin as electron donors. E. coli has three glutaredoxins: 1, 2, and 3, each with a classical -Cys-Pro-Tyr-Cys- active site. Glutaredoxin 2 is the major glutathione disulfide oxidoreductase in E. coli, but its function remains unknown. In this report glutaredoxin 2 is shown to be the most effective hydrogen donor for the reduction of arsenate by ArsC. Analysis of single or double cysteine-to-serine substitutions in the active site of the three glutaredoxins indicated that only the N-terminal cysteine residue is essential for activity. This suggests that, during the catalytic cycle, ArsC forms a mixed disulfide with GSH before being reduced by glutaredoxin to regenerate the active ArsC reductase.

Arsenates↗

PubMed1994

Two additional glutaredoxins exist in Escherichia coli: glutaredoxin 3 is a hydrogen donor for ribonucleotide reductase in a thioredoxin/glutaredoxin 1 double mutant.

Thioredoxin (Trx) and glutaredoxin (Grx1) are hydrogen donors for ribonucleotide reductase, the key enzyme for deoxyribonucleotide biosynthesis. The viability of a double mutant lacking both Trx and Grx1 implies the presence of a third, unknown hydrogen donor. This paper reports the purification and characterization of two proteins with glutaredoxin activity (using hydroxyethyl disulfide as a substrate) from an Escherichia coli mutant lacking Trx and Grx1 (delta trxA, grx::kan). Affinity chromatography was used to bind glutaredoxin on a glutathione-containing thiol-Sepharose column. The molecular weight of Grx2, 27,000, was atypical for glutaredoxins, whereas Grx3 had a molecular weight of 10,000. Amino acid sequence analysis revealed novel structures with putative active sites typical of glutaredoxins: Cys-Pro-Tyr-Cys. The proteins are therefore referred to as Grx2 and Grx3. The low hydrogen donor activity for ribonucleotide reductase in the crude extract was recovered in the purification of Grx3, whereas Grx2 was inactive. As a hydrogen donor for E. coli ribonucleotide reductase, Grx3 showed approximately the same Km value (0.35 microM) as Grx1, whereas its Vmax value was only 5% that of Grx1. The combination of the Grx3 hydrogen donor activity and a 25-fold induction of ribonucleotide reductase activity in a delta trxA, grx double mutant provides an explanation for its viability and deoxyribonucleotide biosynthesis. The physiological functions of Grx2 and Grx3 remain to be determined.

Amino Acid Sequence↗

PubMed2006

Role of glutaredoxin-3 and glutaredoxin-4 in the iron regulation of the Aft1 transcriptional activator in Saccharomyces cerevisiae.

The transcription factors Aft1 and Aft2 from Saccharomyces cerevisiae regulate the expression of genes involved in iron homeostasis. These factors induce the expression of iron regulon genes in iron-deficient yeast but are inactivated in iron-replete cells. Iron inhibition of Aft1/Aft2 was previously shown to be dependent on mitochondrial components required for cytosolic iron sulfur protein biogenesis. We presently show that the nuclear monothiol glutaredoxins Grx3 and Grx4 are critical for iron inhibition of Aft1 in yeast cells. Cells lacking both glutaredoxins show constitutive expression of iron regulon genes. Overexpression of Grx4 attenuates wild type Aft1 activity. The thioredoxin-like domain in Grx3 and Grx4 is dispensable in mediating iron inhibition of Aft1 activity, whereas the conserved cysteine that is part of the conserved CGFS motif in monothiol glutaredoxins is essential for this function. Grx3 and Grx4 interact with Aft1 as shown by two-hybrid interactions and co-immunoprecipitation assays. The interaction between glutaredoxins and Aft1 is not modulated by the iron status of cells but is dependent on the conserved glutaredoxin domain Cys residue. Thus, Grx3 and Grx4 are novel components required for Aft1 iron regulation that most likely occurs in the nucleus.

Cell Nucleus↗

PubMed2005

NMR reveals a novel glutaredoxin-glutaredoxin interaction interface.

Glutaredoxins (Grx) represent a large family of glutathione (GSH)-dependent oxidoreductases that catalyse the reduction of disulfides or glutathione mixed disulfide. Grx domains from pathogenic bacteria and plant Grxs have been recently reported to target specific peroxiredoxins (Prxs). The specificity that triggers the interaction between Grx and Prx is poorly understood and is only based on the structure of Haemophilus influenzae Prx-Grx hybrid (hyPrx5). We report here an NMR study of the Populus tremula Grx C4 that targets a P.tremula D-type II Prx. We show that Grx C4 specifically self-associates in a monomer-dimer equilibrium with an apparent K(d) of ca 2.6 mM. Grx C4 homodimer was docked under experimental restraints. The results reveal a novel Grx-Grx interface that is unrelated to the hyPrx5 Grx-Grx dimer interface. Chemical-shift perturbations and 15N spin-relaxation measurements show that the auto-association surface comprises both the active site and the GSH binding site. Reduced GSH is demonstrated to bind reduced Grx with a K(d) of ca 8.6 mM. The potential biological significance of the new Grx-Grx interaction interface is discussed.

Amino Acid Sequence↗

PubMed2002

Role of glutaredoxin in metabolic oxidative stress. Glutaredoxin as a sensor of oxidative stress mediated by H2O2.

Epitope-tagged glutaredoxin (GRX) was utilized to determine the role of GRX in oxidative stress-induced signaling and cytotoxicity in glucose-deprived human cancer cells (MCF-7/ADR and DU-145). GRX-overexpressing cells demonstrated resistance to glucose deprivation-induced cytotoxicity and decreased activation of c-Jun N-terminal kinase (JNK1). Deletion mutants showed the C-terminal portion of apoptosis signal-regulating kinase 1 (ASK1) bound GRX, and glucose deprivation disrupted binding. Treatment with l-buthionine-(S,R)-sulfoximine reduced glutathione content by 99% and prevented glucose deprivation-induced dissociation of GRX from ASK1. A thiol antioxidant, N-acetyl-l-cysteine, or overexpression of an H(2)O(2) scavenger, catalase, inhibited glucose deprivation-induced dissociation of GRX from ASK1. GRX active site cysteine residues (Cys(22) and Cys(25)) were required for dissociation of GRX from ASK1 during glucose deprivation. Kinase assays revealed that SEK1 and JNK1 were regulated in an ASK1-dependent fashion during glucose deprivation. Overexpression of GRX or catalase inhibited activation of ASK1-SEK1-JNK1 signaling during glucose deprivation. These results demonstrate that GRX is a negative regulator of ASK1 and dissociation of GRX from ASK1 activates ASK1-SEK1-JNK1 signaling leading to cytotoxicity during glucose deprivation. These results support the hypothesis that the GRX-ASK1 interaction is redox sensitive and regulated in a glutathione-dependent fashion by H(2)O(2).

Acetylcysteine↗

PubMed1996

S-glutathiolated hepatocyte proteins and insulin disulfides as substrates for reduction by glutaredoxin, thioredoxin, protein disulfide isomerase, and glutathione.

The disulfide-reducing activities of glutaredoxin, thioredoxin, protein disulfide isomerase, glutathione, and cysteine were directly compared with a mixture of hepatocyte 35S-glutathiolated proteins as the substrate. Dethiolation of individual 35S-labeled protein bands from the mixture of hepatocyte proteins was analyzed by SDS-PAGE. All of the 35S-labeled protein bands could be completely dethiolated by glutaredoxin, thioredoxin, protein disulfide isomerase, glutathione, or cysteine. On a molar basis glutaredoxin was over 10 times more effective than either thioredoxin or protein disulfide isomerase. Dethiolation rates of individual proteins varied in minor ways. For example, glutaredoxin dethiolated the 15-, 30-, and 48-kDa protein bands 3 to 4 times faster than the 27-, 28-, and 77-kDa bands. Glutaredoxins from pig liver or from bovine heart had the same specificity and similar activity. The rate of dethiolation by glutathione alone was low compared to the glutaredoxin-catalyzed process, but all 35S-labeled protein bands could be reduced by glutathione, cysteine, or dithiothreitol. Glutathione was clearly more effective than cysteine when these two thiols were compared on the basis of the concentration of thiolate anion available at neutral pH. Therefore, glutathione is a more specific reductant of S-glutathiolated proteins than is cysteine but it is much less effective than glutaredoxin. Since glutaredoxin activity in cells is 10 times higher than the concentration used in these experiments, ample activity is available to account for substantial rates of dethiolation in vivo. Thioredoxin is quite inefficient as a reductant of S-glutathiolated proteins, but it was reasoned that it might first reduce glutaredoxin, which then could reduce the S-glutathiolated protein. A combination of thioredoxin and glutaredoxin was effective. It is proposed that glutaredoxin is the principal agent responsible for protein dethiolation in vivo. The effectiveness of glutaredoxin, thioredoxin, and protein disulfide isomerase as reductants for protein disulfide bonds was examined with insulin as the substrate. Protein disulfide isomerase was very effective and thioredoxin was nearly as effective. Human thioredoxin was similar to Escherichia coli thioredoxin in reactivity and specificity. Glutaredoxin did not facilitate insulin reduction at equal concentrations. Thus, protein disulfide isomerase and thioredoxin are more effective than glutaredoxin as reductants of insulin protein disulfides. The apparent reduction potential of pig liver glutaredoxin (-0.159 +/- 0.004 V) was determined by measuring the amount of reduced glutaredoxin in equilibrium with mixtures of glutathione and glutathione disulfide. Glutaredoxin was a weaker reductant than E. coli thioredoxin (-0.260 V) and was similar to protein disulfide isomerase (-0.11 to -0.19 V). The role of these proteins as disulfide reductants is not determined solely by thermodynamic considerations. A glutathione binding site at the dithiol region of glutaredoxin may be of primary importance for its function in protein dethiolation, while a different specific peptide binding site in thioredoxin may be more suited to certain protein disulfide structures.

Animals↗

PubMed1995

Purification from placenta, amino acid sequence, structure comparisons and cDNA cloning of human glutaredoxin.

Glutaredoxin is generally a glutathione-dependent hydrogen donor for ribonucleotide reductase and also catalyses general glutathione (GSH)-disulfide-oxidoreduction reactions in the presence of NADPH and glutathione reductase. A Glutaredoxin from human placenta was purified to homogeneity, as judged by SDS/PAGE and IEF (12 kDa). Purification was monitored by the activity with hydroxyethyl disulfide as a substrate. Values of pI for glutaredoxin were obtained by IEF; the pI of the protein shifted from 7.3 in its fully reduced state to 9.0 in the oxidized state after treatment with excess hydroxyethyl disulfide. The glutaredoxin preparation showed GSH-dependent hydrogen-donor activity with recombinant mouse ribonucleotide reductase, it exhibited dehydroascorbate reductase activity as well as hydroxyethyl-disulfide-reducing activity. The amino acid sequence (residues 3-104) of glutaredoxin was determined by peptide sequencing and residues 1, 2 and 105 by cDNA sequence analysis. The glutaredoxin sequence comprised the classical active site for glutaredoxins -Cys22-Pro-Tyr-Cys25- and three additional half-cystine residues; two of these in positions 78 and 82. The sequence was similar to other known mammalian glutaredoxins (about 80% identities), with important differences such as one additional Cys residue (Cys7) and no Met residue. The sequence of human glutaredoxin was compared to that of Escherichia coli glutaredoxin with known three-dimensional structure in solution to identify conserved residues and predict a structure from alignment. In particular the GSH-binding site of glutaredoxin was conserved between all molecules. A cDNA that encodes the entire glutaredoxin gene (grx) and flanking sequences was isolated from a human spleen cDNA library. The nucleotide sequence of this cDNA (0.8 kb) was determined, including the complete grx gene.

Amino Acid Sequence↗

PubMed1983

The primary structure of Escherichia coli glutaredoxin. Distant homology with thioredoxins in a superfamily of small proteins with a redox-active cystine disulfide/cysteine dithiol.

An immunosorbent method using antiglutaredoxin-Sepharose was developed for purification of glutaredoxin in high yield from a mutant strain of Escherichia coli K 12 lacking thioredoxin reductase (C 10-17). The primary structure of the protein was determined by analyses of [14C]carboxymethylated glutaredoxin and its proteolytic fragments obtained by digestions with trypsin, clostripain, chymotrypsin and staphylococcal Glu-specific extracellular protease. The single active-center disulfide has the structure-Cys-Pro-Tyr-Cys-, with the half-cystine residues located at positions 11 and 14 in the polypeptide chain. In total the protein was deduced to have 85 residues corresponding to a molecular weight of 9674 for the reduced form of glutaredoxin, making it one of the smallest known enzymes (a glutathione-disulfide transhydrogenase). The half-cystines are identically spaced and similarly positioned in the N-terminal part of the protein when compared with a corresponding functionally active disulfide/dithiol in thioredoxins. Glutaredoxin is also distantly homologous with thioredoxins from phage T4 and E. coli, but extensive differences, even around the redox-active disulfide, distinguish glutaredoxin from the thioredoxins. Allowing for deletions in the glutaredoxin sequence (or insertions in the T4 thioredoxin sequence) at four places, there are identical residues at 25 positions of the 77 compared (= 32% identity). The results establish that glutaredoxin belongs to the same superfamily of small redox proteins as the thioredoxins. The structures are, however, subject to large changes, only four positions have residues identical among all presently analyzed forms. The fluorescence of reduced and oxidized glutaredoxin demonstrates an increase in the quantum yield of the tyrosine emission upon reduction with dithiothreitol. Differences in the spectra support the presence of tyrosine adjacent to the redox-active disulfide bridge. They also confirm that glutaredoxin lacks the disulfide-adjacent tryptophan residues of E. coli thioredoxin. There are known to be great differences between the bacterial E. coli and phage T4 forms of thioredoxin. The glutaredoxin structure is most similar to the phage type, both with respect to size of the polypeptide chain and to actual sequence. From the structural results and the previously known functional similarities it appears possible that the phage thioredoxin may have evolved from an early glutaredoxin gene. The mixed properties are compatible with this conclusion, the superfamily assignment, and the differences in biological activity.

Amino Acid Sequence↗

PubMed1993

Immunochemical characterization and tissue distribution of glutaredoxin (thioltransferase) from calf.

Glutaredoxin catalyzes glutathione-dependent disulfide oxidoreduction reactions in a coupled system with NADPH, GSH and glutathione reductase and has an active site disulfide/dithiol with the sequence -Cys-Pro-Tyr-Cys-. Calf thymus glutaredoxin (thioltransferase), which contains two additional structural half-cystine residues, was purified to homogeneity, using a modification of the previously described isolation procedure. This method involved a pI-shift of glutaredoxin, obtained after oxidation of the fully reduced form with hydroxyethyl-disulfide, followed by CM-Sepharose chromatography. On both SDS- and IEF-gels the protein migrated as one band (M(r) 12,000). The pure protein was used to affinity-purify rabbit antiglutaredoxin antibodies obtained by immunization with the oxidized form of glutaredoxin. Using these antibodies the distribution of glutaredoxin was mapped in calf organs and tissues by Western blots and by immunohistochemistry. Glutaredoxin was demonstrated in all organs investigated. Western blots showed the presence of weak additional high molecular weight bands of unknown identity in certain organs. The immunohistochemical analyses revealed that glutaredoxin is highly expressed in a wide variety of cell types, both epithelial and mesenchymal. The distribution and occurrence in the calf organs was similar to that previously described for thioredoxin in the rat. There were some exceptions: e.g., follicular cells in the ovary did not contain immunohistochemically demonstrable glutaredoxin but expressed thioredoxin. Particularly striking were observations of strong glutaredoxin immunoreactivity in oocytes in the ovary and the pattern of glutaredoxin in epithelial tissue of the skin and tongue reflecting differential expression during cell differentiation. The distribution demonstrated that glutaredoxin serves functions apart from the originally described role as hydrogen donor for ribonucleotide reductase which only occurs in replicating cells. Such functions should relate particularly to glutathione-catalyzed protein disulfide oxidoreductions and cellular signalling by redox regulating mechanisms.

Amino Acid Sequence↗

PubMed1991

Escherichia coli glutaredoxin: cloning and overexpression, thermodynamic stability of the oxidized and reduced forms, and report of an N-terminal extended species.

Escherichia coli glutaredoxin (MW 9700) catalyzes intracellular redox reactions utilizing a disulfide/dithiol enzymatic mechanism involving the active-site residues -Cys-Pro-Tyr-Cys-. It is functionally related to the thioredoxin family and is expected to share similar three-dimensional structure [Eklund, H., Cambillau, C., Sjöberg, B.-M., Holmgren, A., Jörnvall, H., Höög, J.-O., & Brändén, C.-I. (1984) EMBO J. 3, 1443-1449]. We constructed an overexpression system in which production of glutaredoxin is controlled by temperature-sensitive expression of the phage T7 promoter. In addition to glutaredoxin, a second gene product is observed; this species, which we call glutaredoxin N, is glutaredoxin extended by the sequence Met-Arg-Arg-Glu-Ile- at the N terminus. We have begun characterization of the structure and stability of the oxidized and reduced forms of glutaredoxin (grx-S2 and grx-(SH)2, respectively). Secondary structure calculated from CD data agrees with that predicted from the three-dimensional model of Eklund et al. The cooperative denaturation reactions of oxidized and reduced glutaredoxin were measured in temperature-induced and guanidine hydrochloride induced unfolding experiments. Surprisingly, oxidized and reduced glutaredoxins are very similar in stability. In heat-induced denaturation, monitored by CD, Tm is 55 and 57 degrees C for oxidized and reduced, respectively. In GuHCl denaturation, monitored by fluorescence, the midpoint denaturant concentrations are 2 M for both oxidized and reduced. It follows that the redox potentials of the disulfide bond are similar in unfolded and folded glutaredoxin. This is unexpected because in E. coli thioredoxin the oxidized form is far more stable than the reduced [Kelley, R.F., Shalongo, W., Jagannadham, M.V., & Stellwagen, E. (1987) Biochemistry 26, 1406-1411] and the redox potential of folded thioredoxin is significantly more negative than that of unfolded thioredoxin [Lin, T.-Y., & Kim, P. (1989) Biochemistry 28, 5282-5287].

Amino Acid Sequence↗

PubMed2006

Selective inactivation of glutaredoxin by sporidesmin and other epidithiopiperazinediones.

Glutaredoxin (thioltransferase) is a thiol-disulfide oxidoreductase that displays efficient and specific catalysis of protein-SSG deglutathionylation and is thereby implicated in homeostatic regulation of the thiol-disulfide status of cellular proteins. Sporidesmin is an epidithiopiperazine-2,5-dione (ETP) fungal toxin that disrupts cellular functions likely via oxidative alteration of cysteine residues on key proteins. In the current study sporidesmin inactivated human glutaredoxin in a time- and concentration-dependent manner. Under comparable conditions other thiol-disulfide oxidoreductase enzymes, glutathione reductase, thioredoxin, and thioredoxin reductase, were unaffected by sporidesmin. Inactivation of glutaredoxin required the reduced (dithiol) form of the enzyme, the oxidized (intramolecular disulfide) form of sporidesmin, and molecular oxygen. The inactivated glutaredoxin could be reactivated by dithiothreitol only in the presence of urea, followed by removal of the denaturant, indicating that inactivation of the enzyme involves a conformationally inaccessible disulfide bond(s). Various cysteine-to-serine mutants of glutaredoxin were resistant to inactivation by sporidesmin, suggesting that the inactivation reaction specifically involves at least two of the five cysteine residues in human glutaredoxin. The relative ability of various epidithiopiperazine-2,5-diones to inactivate glutaredoxin indicated that at least one phenyl substituent was required in addition to the epidithiodioxopiperazine moiety for inhibitory activity. Mass spectrometry of the modified protein is consistent with formation of intermolecular disulfides, containing one adducted toxin per glutaredoxin but with elimination of two sulfur atoms from the detected product. We suggest that the initial reaction is between the toxin sulfurs and cysteine 22 in the glutaredoxin active site. This study implicates selective modification of sulfhydryls of target proteins in some of the cytotoxic effects of the ETP fungal toxins and their synthetic analogues.

Amino Acid Substitution↗

PubMed2005

Molecular mapping of functionalities in the solution structure of reduced Grx4, a monothiol glutaredoxin from Escherichia coli.

The ubiquitous glutaredoxin protein family is present in both prokaryotes and eukaryotes, and is closely related to the thioredoxins, which reduce their substrates using a dithiol mechanism as part of the cellular defense against oxidative stress. Recently identified monothiol glutaredoxins, which must use a different functional mechanism, appear to be essential in both Escherichia coli and yeast and are well conserved in higher order genomes. We have employed high resolution NMR to determine the three-dimensional solution structure of a monothiol glutaredoxin, the reduced E. coli Grx4. The Grx4 structure comprises a glutaredoxin-like alpha-beta fold, founded on a limited set of strictly conserved and structurally critical residues. A tight hydrophobic core, together with a stringent set of secondary structure elements, is thus likely to be present in all monothiol glutaredoxins. A set of exposed and conserved residues form a surface region, implied in glutathione binding from a known structure of E. coli Grx3. The absence of glutaredoxin activity in E. coli Grx4 can be understood based on small but significant differences in the glutathione binding region, and through the lack of a conserved second GSH binding site. MALDI experiments suggest that disulfide formation on glutathionylation is accompanied by significant structural changes, in contrast with dithiol thioredoxins and glutaredoxins, where differences between oxidized and reduced forms are subtle and local. Structural and functional implications are discussed with particular emphasis on identifying common monothiol glutaredoxin properties in substrate specificity and ligand binding events, linking the thioredoxin and glutaredoxin systems.

Amino Acid Motifs↗

PubMed2006

Insights into deglutathionylation reactions. Different intermediates in the glutaredoxin and protein disulfide isomerase catalyzed reactions are defined by the gamma-linkage present in glutathione.

Glutaredoxins are small proteins with a conserved active site (-CXX(C/S)-) and thioredoxin fold. These thiol disulfide oxidoreductases catalyze disulfide reductions, preferring GSH-mixed disulfides as substrates. We have developed a new real-time fluorescence-based method for measuring the deglutathionylation activity of glutaredoxins using a glutathionylated peptide as a substrate. Mass spectrometric analysis showed that the only intermediate in the reaction is the glutaredoxin-GSH mixed disulfide. This specificity was solely dependent on the unusual gamma-linkage present in glutathione. The deglutathionylation activity of both wild-type Escherichia coli glutaredoxin and the C14S mutant was competitively inhibited by oxidized glutathione, with K(i) values similar to the K(m) values for the glutathionylated peptide substrate, implying that glutaredoxin primarily recognizes the substrate via the glutathione moiety. In addition, wild-type glutaredoxin showed a sigmoidal dependence on GSH concentrations, the activity being significantly decreased at low GSH concentrations. Thus, under oxidative stress conditions, where the ratio of GSH/GSSG is decreased, the activity of glutaredoxin is dramatically reduced, and it will only have significant deglutathionylation activity once the oxidative stress has been removed. Different members of the protein disulfide isomerases (PDI) family showed lower activity levels when compared with glutaredoxins; however, their deglutathionylation activities were comparable with their oxidase activities. Furthermore, in contrast to the glutaredoxin-GSH mixed disulfide intermediate, the only intermediate in the PDI-catalyzed reaction was PDI peptide mixed disulfide.

Catalysis↗

PubMed1997

Complete 1H, 13C, and 15N NMR resonance assignments and secondary structure of human glutaredoxin in the fully reduced form.

Human glutaredoxin is a member of the glutaredoxin family, which is characterized by a glutathione binding site and a redox-active dithiol/disulfide in the active site. Unlike Escherichia coli glutaredoxin-1, this protein has additional cysteine residues that have been suggested to play a regulatory role in its activity. Human glutaredoxin (106 amino acid residues, M(r) = 12,000) has been purified from a pET expression vector with both uniform 15N labeling and 13C/15N double labeling. The combination of three-dimensional 15N-edited TOCSY, 15N-edited NOESY, HNCA, HN(CO)CA, and gradient sensitivity-enhanced HNCACB and HNCO spectra were used to obtain sequential assignments for residues 2-106 of the protein. The gradient-enhanced version of the HCCH-TOCSY pulse sequence and HCCH-COSY were used to obtain side chain 1H and 13C assignments. The secondary structural elements in the reduced protein were identified based on NOE information, amide proton exchange data, and chemical shift index data. Human glutaredoxin contains five helices extending approximately from residues 4-10, 24-36, 53-64, 83-92, and 94-104. The secondary structure also shows four beta-strands comprised of residues 15-19, 43-48, 71-75, 78-80, which form a beta-sheet almost identical to that found in E. coli glutaredoxin-1. Complete 1H, 13C, and 15N assignments and the secondary structure of fully reduced human glutaredoxin are presented. Comparison to the structures of other glutaredoxins is presented and differences in the secondary structure elements are discussed.

Amino Acid Sequence↗