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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↗
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↗
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↗
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↗
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↗
PubMed1991

Characterization of homogeneous recombinant glutaredoxin from Escherichia coli: purification from an inducible lambda PL expression system and properties of a novel elongated form.

We have constructed a plasmid, pAHOB1, with a 482-b AluI fragment containing the Escherichia coli glutaredoxin gene (grx) cloned under lambda PL promoter control. Growth of E. coli N4830/pAHOB1 cells at 30 degrees C followed by heat induction at 40 degrees C for 5 h resulted in expression of glutaredoxin as 20% of the soluble E. coli protein. Methods for the preparation of gram amounts of glutaredoxin and 5 mM solutions suitable for NMR studies were developed. About 10% of the glutaredoxin activity showed an unexpected higher isoelectric point and was isolated by DEAE-cellulose chromatography at pH 6.0. Sequence analysis demonstrated that this novel form (grx-90) contained five additional N-terminal residues (Met-Arg-Arg-Glu-Ile) added to the glutaredoxin molecule with 85 residues (grx-85). Grx-90 originates from an alternative ATG initiation codon present 5' of the previously identified translation start site on the grx gene in E. coli. Despite the highly charged N-terminal extension, grx-90 showed full activity as a GSH-disulfide oxidoreductase and the same apparent Km value (0.14 microM) as glutaredoxin in GSH-dependent reduction of CDP by ribonucleotide reductase. Both grx-90 and grx-85 showed identical competition curves in radioimmunoassays. The presence of grx-90 was also demonstrated in log-phase E. coli C600 cells as 5 to 10% of total glutaredoxin by immunological techniques. The molar extinction coefficient of native glutaredoxin (12,500 M-1 cm-1 at 280 nm) was 15% higher than expected from its content of one Trp and four Tyr residues.

Amino Acid Sequence↗
PubMed1996

Purification, cloning and expression of dehydroascorbic acid-reducing activity from human neutrophils: identification as glutaredoxin.

Dehydroascorbic acid-reducing activity in normal human neutrophil lysates was characterized and identified by activity-based purification and measurement of newly synthesized ascorbate by HPLC. The initial reducing activity was non-dialysable and could not be accounted for by the activity of glutathione as a reducing agent. The reducing activity was purified to homogeneity as an 11 kDa protein. The protein had a specific activity of 3 mumol/min per mg of protein and was glutathione dependent. Kinetic experiments showed that the protein had a K(m) for glutathione of 2.0 mM and a K(m) for dehydroascorbic acid of 250 microM. Dehydroascorbic acid reduction by the purified protein was pH dependent and was maximal at pH 7.5. Peptide fragments from the purified protein were analysed for amino acid sequence and the protein was identified as glutaredoxin. By using degenerate oligonucleotides based on the amino acid sequence, glutaredoxin was cloned from a human neutrophil library. Expressed purified glutaredoxin displayed reducing activity and kinetics that were indistinguishable from those of native purified enzyme. Several approaches indicated that glutaredoxin was responsible for the most of the protein-mediated dehydroascorbic acid reduction in lysates. From protein purification data, glutaredoxin was responsible for at least 47% of the initial reducing activity. Dehydroascorbic acid reduction was at least 5-fold greater in neutrophil lysates than in myeloid tumour cell lysates, and glutaredoxin was detected in normal neutrophil lysates but not in myeloid tumour cell lysates by Western blotting. Glutaredoxin inhibitors inhibited dehydroascorbic acid reduction in neutrophil lysates as much as 80%. These findings indicate that glutaredoxin plays a major role in dehydroascorbic acid reduction in normal human neutrophil lysates, and represent the first identification of dehydroascorbic acid reductase in human tissue by activity-based purification.

Amino Acid Sequence↗
PubMed1992

Structural and functional characterization of the mutant Escherichia coli glutaredoxin (C14----S) and its mixed disulfide with glutathione.

Glutaredoxin is essential for the glutathione (GSH)-dependent synthesis of deoxyribonucleotides by ribonucleotide reductase, and in addition, it displays a general GSH disulfide oxidoreductase activity. In Escherichia coli glutaredoxin, the active site contains a redox-active disulfide/dithiol of the sequence Cys11-Pro12-Tyr13-Cys14. In this paper, we have prepared and characterized the Cys14----Ser mutant of E. coli glutaredoxin and its mixed disulfide with glutathione. The Cys14----Ser mutant of glutaredoxin is shown to retain 38% of the GSH disulfide oxidoreductase activity of the wild-type protein with hydroxyethyl disulfide as substrate but to be completely inactive with ribonucleotide reductase, demonstrating that dithiol glutaredoxin is the hydrogen donor for ribonucleotide reductase. The covalent structure of the mixed disulfide of glutaredoxin(C14S) with GSH prepared with 15N-labeling of the protein was confirmed with nuclear magnetic resonance (NMR) spectroscopy, establishing a basis for NMR structural studies of the glutathione binding site on glutaredoxin.

Amino Acid Sequence↗
PubMed1988

Construction and characterization of glutaredoxin-negative mutants of Escherichia coli.

Deoxyribonucleotides, the precursors of DNA, are formed de novo by ribonucleotide reductase, and in vitro thioredoxin or glutathione plus glutaredoxin have been isolated as hydrogen donors. The in vivo hydrogen donor for ribonucleotide reductase is not known. To study this, the Escherichia coli glutaredoxin gene (255 base pairs) was inactivated by inserting a 2-kilobase kanamycin-resistance fragment into the coding sequence of the cloned gene. The inactivated gene was inserted into the E. coli chromosome and mapped to about 18.5 min. A gene replacement technique was used to obtain a strain, A407, that lacked glutaredoxin by radioimmunoassay and by enzymatic assay with ribonucleotide reductase. Glutaredoxin was found not to be essential for viability of E. coli. Thioredoxin is also not essential for viability, as had been shown earlier, but a double mutant lacking glutaredoxin and thioredoxin could not be obtained by P1 transduction on a defined medium, indicating that either thioredoxin or glutaredoxin is essential. In rich medium, very slowly growing, unstable transductants were obtained that at high frequency gave rise to better growing cells. One such isolate, A410, was shown to still lack glutaredoxin and thioredoxin.

Bacterial Proteins↗
PubMed1992

Structure of oxidized bacteriophage T4 glutaredoxin (thioredoxin). Refinement of native and mutant proteins.

The structure of wild-type bacteriophage T4 glutaredoxin (earlier called thioredoxin) in its oxidized form has been refined in a monoclinic crystal form at 2.0 A resolution to a crystallographic R-factor of 0.209. A mutant T4 glutaredoxin gives orthorhombic crystals of better quality. The structure of this mutant has been solved by molecular replacement methods and refined at 1.45 A to an R-value of 0.175. In this mutant glutaredoxin, the active site residues Val15 and Tyr16 have been substituted by Gly and Pro, respectively, to mimic that of Escherichia coli thioredoxin. The main-chain conformation of the wild-type protein is similar in the two independently determined molecules in the asymmetric unit of the monoclinic crystals. On the other hand, side-chain conformations differ considerably between the two molecules due to heterologous packing interactions in the crystals. The structure of the mutant protein is very similar to the wild-type protein, except at mutated positions and at parts involved in crystal contacts. The active site disulfide bridge between Cys14 and Cys17 is located at the first turn of helix alpha 1. The torsion angles of these residues are similar to those of Escherichia coli thioredoxin. The torsion angle around the S-S bond is smaller than that normally observed for disulfides: 58 degrees, 67 degrees and 67 degrees for wild-type glutaredoxin molecule A and B and mutant glutaredoxin, respectively. Each sulfur atom of the disulfide cysteines in T4 glutaredoxin forms a hydrogen bond to one main-chain nitrogen atom. The active site is shielded from solvent on one side by the beta-carbon atoms of the cysteine residues plus side-chains of residues 7, 9, 21 and 33. From the opposite side, there is a cleft where the sulfur atom of Cys14 is accessible and can be attacked by a nucleophilic thiolate ion in the initial step of the reduction reaction.

Amino Acid Sequence↗
PubMed1997

The human glutaredoxin gene: determination of its organization, transcription start point, and promoter analysis.

A genomic clone for the human glutaredoxin gene was isolated and sequenced. An intron was located within the coding region and began 211 nt downstream of the initiator codon. Except for this intron, the genomic sequence shares 100% identity to the published glutaredoxin cDNA sequence. A second intron was located in the 3' UTR 6 bp downstream of the terminator codon. The tsp of the glutaredoxin gene was determined by primer extension and confirmed by S1 mapping analysis. Analysis of the 5'-flanking region of the gene revealed that the promoter sequences TATA and CCAAT were 30 and 160 bp upstream, respectively, from the tsp. Other potential transcription factor binding sites included NF-E1, HNF-5, P2II and AP-1. Glutaredoxin promoter constructs inserted into a reporter plasmid for firefly luciferase were transfected into fibroblasts, and luciferase activity was 8-10-fold higher compared with controls lacking glutaredoxin promoter. These data indicate that the promoter region of the isolated glutaredoxin gene is functional.

Amino Acid Sequence↗
PubMed1997

Characterization of Escherichia coli NrdH. A glutaredoxin-like protein with a thioredoxin-like activity profile.

Ribonucleotides are converted to deoxyribonucleotides by ribonucleotide reductases. Either thioredoxin or glutaredoxin is a required electron donor for class I and II enzymes. Glutaredoxins are reduced by glutathione, thioredoxins by thioredoxin reductase. Recently, a glutaredoxin-like protein, NrdH, was isolated as the functional electron donor for a NrdEF ribonucleotide reductase, a class Ib enzyme, from Lactococcus lactis. The absence of glutathione in this bacterium raised the question of the identity of the intracellular reductant for NrdH. Homologues of NrdH are present in the genomes of Escherichia coli and Salmonella typhimurium, upstream of the genes for the poorly transcribed nrdEF, separated from it by an open reading frame (nrdI) coding for a protein of unknown function. Overexpression of E. coli NrdH protein shows that it is a functional hydrogen donor with higher specificity for the class Ib (NrdEF) than for the class Ia (NrdAB) ribonucleotide reductase. Furthermore, this glutaredoxin-like enzyme is reduced by thioredoxin reductase and not by glutathione. We suggest that several uncharacterized glutaredoxin-like proteins present in the genomes of organisms lacking GSH, including archae, will also react with thioredoxin reductase and be related to the ancestors from which the GSH-dependent glutaredoxins have evolved by the acquisition of a GSH-binding site. We also show that NrdI, encoded by all nrdEF operons, has a stimulatory effect on ribonucleotide reduction.

Amino Acid Sequence↗
PubMed1997

Purification and characterization of glutaredoxin (thioltransferase) from rice (Oryza sativa L.).

We purified and characterized glutaredoxin (thioltransferase), which catalyzes thiol/disulfide exchange reaction, for the first time in plants. The purification procedure employed an immunoabsorbent, antiglutaredoxin-Sepharose. Glutaredoxin was purified about 2,200-fold from rice bran and it appeared to be homogeneous on SDS-PAGE. MALDI-TOF mass spectrometry revealed that the protein has a molecular mass of 11,097.9 Da. Rice glutaredoxin consists of 105 amino acid residues, containing the tetrapeptide -Cys-Phe-Pro (Tyr)-Cys-, which constitutes the active site of Escherichia coli and mammalian glutaredoxins. Inactivation assay also indicated that cysteine residues are responsible for enzyme activity. Kinetic analyses revealed that the enzyme did not exhibit normal Michaelis-Menten kinetics. The enzyme has an optimum pH of about 8.7 with 2-hydroxyethyl disulfide as a substrate. In addition, rice glutaredoxin has dehydroascorbate reductase activity, like mammalian glutaredoxin.

Amino Acid Sequence↗
PubMed1984

The primary structure of calf thymus glutaredoxin. Homology with the corresponding Escherichia coli protein but elongation at both ends and with an additional half-cystine/cysteine pair.

The primary structure of calf thymus glutaredoxin was determined by analysis of the [14C]carboxymethylated protein and the proteolytic fragments obtained by treatments with trypsin, chymotrypsin, CNBr and staphylococcal Glu-specific extracellular protease. The active center has the structure Cys-Pro-Tyr-Cys, with the redox-active cysteines/half-cystines located at positions 22 and 25 in the polypeptide chain. This active center is identical in amino acid sequence and similar in position to that of Escherichia coli glutaredoxin, suggesting this structure to be typical for glutaredoxins and distinguishing them from the distantly related thioredoxins. However, the two glutaredoxins also exhibit considerable differences. Calf thymus glutaredoxin is extended at both ends and has 31% overall residue identities with the corresponding E. coli protein. In contrast to the bacterial glutaredoxin, the calf thymus protein contains two additional half-cystines/cysteine residues at positions 74 and 78, which may be of regulatory significance.

Amino Acid Sequence↗