Hydrogen Donor For Ribonucleotide Reductase Research Articles

Characterization of Human Cytosolic Thioredoxin Mutants with Increasing Side Chain Volumes at Residue-33

E-mail: hjkim@daegu.ac.kr Received June 12, 2012, Accepted July 3, 2012Key Words : Thioredoxin, Thioredoxin reductase, Site-directed mutagenesis Thioredoxin (Trx) is a small protein that exists in mostliving organisms, ranging from bacteria to mammals, andacts as a hydrogen donor for ribonucleotide reductase inEscherichia coli.

Thioredoxin, the Processivity Factor, Sequesters an Exposed Cysteine in the Thumb Domain of Bacteriophage T7 DNA Polymerase

Gene 5 protein (gp5) of bacteriophage T7 is a non-processive DNA polymerase. It achieves processivity by binding to Escherichia coli thioredoxin (trx). gp5/trx complex binds tightly to a primer-DNA template enabling the polymerization of hundreds of nucleotides per binding event. gp5 contains 10 cysteines. Under non-reducing condition, exposed cysteines form intermolecular disulfide linkages resulting in the loss of polymerase activity. No disulfide linkage is detected when Cys-275 and Cys-313 are replaced with serines. Cys-275 and Cys-313 are located on loop A and loop B of the thioredoxin binding domain, respectively. Replacement of either cysteine with serine (gp5-C275S, gp5-C313S) drastically decreases polymerase activity of gp5 on dA(350)/dT(25). On this primer-template gp5/trx in which Cys-313 or Cys-275 is replaced with serine have 50 and 90%, respectively, of the polymerase activity observed with wild-type gp5/trx. With single-stranded M13 DNA as a template gp5-C275S/trx retains 60% of the polymerase activity of wild-type gp5/trx. In contrast, gp5-C313S/trx has only one-tenth of the polymerase activity of wild-type gp5/trx on M13 DNA. Both wild-type gp5/trx and gp5-C275S/trx catalyze the synthesis of the entire complementary strand of M13 DNA, whereas gp5-C313S/trx has difficulty in synthesizing DNA through sites of secondary structure. gp5-C313S fails to form a functional complex with trx as measured by the apparent binding affinity as well as by the lack of a physical interaction with thioredoxin during hydroxyapatite-phosphate chromatography. Small angle x-ray scattering reveals an elongated conformation of gp5-C313S in comparison to a compact and spherical conformation of wild-type gp5.

Regulation of the ribonucleotide reductases inLactococcus lactissubsp.cremoris

Lactococcus lactis has two essential ribonucleotide reductases for DNA biosynthesis and repair which are affected in the presence or absence of oxygen. Expression of glutaredoxin like protein (NrdH), the hydrogen donor for ribonucleotide reductase, was found to be regulated by the FNR like proteins (FlpA and FlpB). Proteomics study demonstrated that expression level of NrdH significantly decreased in the flpA and flpAB deletion mutants. The nrdH gene is located in an nrdHIEF operon and encoding the NrdEF ribonucleotide reductase, which is active under aerobic and anaerobic conditions. Regulation of expression of the nrdHIEF operons was investigated using beta-galactosidase as a reporter gene. The 588 bp fragment containing the nrdH promoter and gene cloned into the pORI vector immediately upstream of a promoterless lacZ gene. Constructed plasmid was transferred into wild type (MG1363), single mutant (flpA orflpB) and double mutant (flpAB). Aerobically, nrdH promoter activity is 15-fold higher than anaerobic expression.

Non-reciprocal regulation of the redox state of the glutathione-glutaredoxin and thioredoxin systems.

Our studies in yeast show that there is an essential requirement for either an active thioredoxin or an active glutathione (GSH)-glutaredoxin system for cell viability. Glutathione reductase (Glr1) and thioredoxin reductase (Trr1) are key regulatory enzymes that determine the redox state of the GSH-glutaredoxin and thioredoxin systems, respectively. Here we show that Trr1 is required during normal cell growth, whereas there is no apparent requirement for Glr1. Analysis of the redox state of thioredoxins and glutaredoxins in glr1 and trr1 mutants reveals that thioredoxins are maintained independently of the glutathione system. In contrast, there is a strong correlation between the redox state of glutaredoxins and the oxidation state of the GSSG/2GSH redox couple. We suggest that independent redox regulation of thioredoxins enables cells to survive in conditions under which the GSH-glutaredoxin system is oxidized.

Immunohistochemical determination of thioredoxin and glutaredoxin distribution in the human cervix ,and possible relation to cervical ripening

Thioredoxin (Trx) and glutaredoxin (Grx) are dithiol redox enzymes ,catalyzing general thiol-disulfide oxidoreductions apart from being hydrogen donors for ribonucleotide reductase ,an enzyme essentialfor DNA synthesis. In mammals ,isoenzymes of Trx and Grx are found in the cytoplasm (Trx1 and Grx1) or in mitochondria (Trx2 and Grx2). Trx and Grx play a role in cellular defence against oxidative stressand in redox regulation of cellular function.The localization and levels of human Trx1 and human Grx1 have been determined in the human cervix by immunohistochemistry and image analysis. Cervical biopsieswere obtained from five non-pregnant ,five term pregnant and five postpartum women. The levels of both Trx1 and Grx1 were increased in the nuclei (after translocation from the cytoplasm) of stromal cellsin cervices from the term pregnant group as compared to the non-pregnant group ,but the levels in the postpartum group did not differ significantly from those of the other two groups. These results arein agreement with our previous data on the mRNA expression of these two redox enzymes. The increased levels of the redox enzymes in term pregnancy suggest that they can be regulating factors involved inthe process of cervical ripening ,e.g. transcription factors and enzymes. Secreted Trx may participate in removing inhibitors of collagen-degrading metalloproteinases.

Characterization of Escherichia coli Null Mutants for Glutaredoxin 2

Three Escherichia coli glutaredoxins catalyze GSH-disulfide oxidoreductions, but the atypical 24-kDa glutaredoxin 2 (Grx2, grxB gene), in contrast to the 9-kDa glutaredoxin 1 (Grx1, grxA gene) and glutaredoxin 3 (Grx3, grxC gene), is not a hydrogen donor for ribonucleotide reductase. To improve the understanding of glutaredoxin function, a null mutant for grxB (grxB(-)) was constructed and combined with other mutations. Null mutants for grxB or all three glutaredoxin genes were viable in rich and minimal media with little changes in their growth properties. Expression of leaderless alkaline phosphatase showed that Grx1 and Grx2 (but not Grx3) contributed in the reduction of cytosolic protein disulfides. Moreover, Grx1 could catalyze disulfide formation in the oxidizing cytosol of combined null mutants for glutathione reductase and thioredoxin 1. grxB(-) cells were more sensitive to hydrogen peroxide and other oxidants and showed increased carbonylation of intracellular proteins, particularly in the stationary phase. Significant up-regulation of catalase activity was observed in null mutants for thioredoxin 1 and the three glutaredoxins, whereas up-regulation of glutaredoxin activity was observed in catalase-deficient strains with additional defects in the thioredoxin pathway. The expression of catalases is thus interconnected with the thioredoxin/glutaredoxin pathways in the antioxidant response.

Cloning and Expression of a Novel Human Glutaredoxin (Grx2) with Mitochondrial and Nuclear Isoforms

Glutaredoxin (Grx) is a glutathione-dependent hydrogen donor for ribonucleotide reductase. Today glutaredoxins are known as a multifunctional family of GSH-disulfide-oxidoreductases belonging to the thioredoxin fold superfamily. In contrast to Escherichia coli and yeast, a single human glutaredoxin is known. We have identified and cloned a novel 18-kDa human dithiol glutaredoxin, named glutaredoxin-2 (Grx2), which is 34% identical to the previously known cytosolic 12-kDa human Grx1. The human Grx2 sequence contains three characteristic regions of the glutaredoxin family: the dithiol/disulfide active site, CSYC, the GSH binding site, and a hydrophobic surface area. The human Grx2 gene, located at chromosome 1q31.2--31.3, consisted of five exons that were transcribed to a 0.9-kilobase human Grx2 mRNA ubiquitously expressed in several tissues. Two alternatively spliced Grx2 mRNA isoforms that differed in their 5' region were identified. These corresponded to alternative proteins with a common 125-residue C-terminal Grx domain but with different N-terminal extensions of 39 and 40 residues, respectively. The 125-residue Grx domain and the two full-length variants were expressed in E. coli and exhibited GSH-dependent hydroxyethyl disulfide and dehydroascorbate reducing activities. Western blot analysis of subcellular fractions from Jurkat cells with a specific anti-Grx2 antibody showed that human Grx2 was predominantly located in the nucleus but also present in the mitochondria. We further showed that one of the mRNA isoforms corresponding to Grx2a encoded a functional N-terminal mitochondrial translocation signal.

Thioredoxin expression in human myometrium and fibroids.

Thioredoxin is a small multifunctional protein which acts as a dithiol hydrogen donor for ribonucleotide reductase in DNA synthesis. Thioredoxin participates in the regulation of different metabolic processes, such as changes in the activity of different enzymes, receptors or transcription factors. The aim of the present study was to determine possible differences in the expression of thioredoxin between myometrium and fibroids in women during different periods of life. Thioredoxin mRNA concentrations were determined in myometrial and fibroid tissues obtained from women during the menstrual cycle, during treatment with an analogue of gonadotrophin releasing hormone (GnRH agonist), in the postmenopausal period (PMP) and during pregnancy. The concentration of thioredoxin mRNA was measured by a solution hybridization method. The localization of thioredoxin protein was examined by immunohistochemistry. There were significantly lower levels of thioredoxin expression in both fibroids and myometrium from GnRH agonist treated and PMP women in comparison with the pregnant women. No difference in thioredoxin expression was found between myometrium and fibroids from the same woman or between myometria from uteri with or without fibroids in the same patient group. Thioredoxin expression in uterine fibroids does not seem to be up-regulated, but changes in response to the endocrine conditions in a similar way to that observed in the myometrium.

Assignment of 1H, 13C, and 15N resonances of reduced Escherichia coli glutaredoxin 2.

The thioredoxin and glutaredoxin families participate as hydrogen donors in a wide variety of cellular reactions (Holmgren, 1989). Glutaredoxins are distinguished from thioredoxins by their ability to catalyze glutathione (GSH)-disulfide oxidoreductions via two redox-active cysteine residues (Holmgren and Aslund, 1995). Glutaredoxin 2 (Grx2) is one of the three glutaredoxins found in E. coli, but unlike the other two, Grx2 is not a hydrogen donor for ribonucleotide reductase (Aslund et al., 1994). It has an unusually large size (24.3 kDa) with 215 amino acid residues, whereas the typical molecular weights for glutaredoxins are around 10 kDa. Grx2 has little sequence homology with other glutaredoxins or thioredoxins, except it contains the conserved active site sequence of Cys-Pro-Tyr-Cys. Grx2 appears most abundant among all three glutaredoxins in E. coli. It may account for about 80% of the glutaredoxin activity in the GSH-disulfide oxidoreductase assay (Vlamis-Gardikas et al., 1997). Although the specific biological function is still unknown, the unique properties of Grx2 indicate that it is likely to have a distinctive function. Grx2 has been overproduced in E. coli; the protein was doubly labeled with 13C and 15N, and triply labeled with 2H, 13C and 15N. Multidimensional NMR spectroscopic studies of the labeled Grx2 have led to extensive backbone and side-chain resonance assignments.

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.

The Levels of Ribonucleotide Reductase, Thioredoxin, Glutaredoxin 1, and GSH Are Balanced in Escherichia coli K12

The dithiol forms of thioredoxin and glutaredoxin are hydrogen donors for ribonucleotide reductase. We have determined the intracellular levels of ribonucleotide reductase (RRase), thioredoxin (Trx), glutaredoxin 1 (Grx1), and glutathione (GSH) and the glutathione redox status in new Escherichia coli K12 strains lacking thioredoxin (trxA-), glutaredoxin 1 (grxA-), and/or GSH (gshA-) or overproducing Trx or Grx1 from multicopy plasmids. We propose a regulatory network in which RRase levels are balanced with those of Trx, Grx1, and GSH so that deficiency or overproduction of one component would promote the opposite effect on the others to maintain a balanced supply of deoxyribonucleotides. GSH deficiency strongly increased both Grx1 levels and RRase activity, even more than Trx deficiency. Double gshA-trxA- bacteria were viable, whereas additional deficiency in lipoate synthesis (gshA-trxA-lipA-) caused the inability to grow in minimal medium plates supplemented with acetate plus succinate instead of lipoic acid. Thus, lipoate might be the only substitute of GSH for glutaredoxin reduction in gshA-trxA- cells, although the extremely high Grx1 content (55-fold) of these bacteria suggests that electron transfer from lipoate might be an inefficient reduction mechanism of glutaredoxins. Moreover, the enhanced Grx1 level of gshA-trxA- cells could obviate the need for a large increase in RRase activity, in contrast to grxA-trxA- double mutant cells. Impairment of the sulfate assimilation pathway, leading to very low GSH concentrations, and an oxidized glutathione redox state might explain the inability of grxA-trxA- cells to grow in minimal medium. Restoration of nearly normal levels of both GSH content and redox status cure the growth defect.

Identification of a Second Functional Glutaredoxin Encoded by the Bacteriophage T4 Genome

Thioredoxins and glutaredoxins are small ubiquitous redox proteins that were discovered as hydrogen donors for ribonucleotide reductase, the key enzyme for deoxyribonucleotide biosynthesis. Some organisms encode more than one redox protein. In this study, we demonstrate that an open reading frame in the bacteriophage T4 genome, reported earlier and designated as Y55.7 (Tomaschewski, J., and Rüger, W. (1987) Nucleic Acids Res. 15, 3632-3633), encodes a second functional redox protein. Gene y55.7 was cloned and expressed in Escherichia coli. Purified Y55.7 protein had glutathione-dependent thioltransferase and dehydroascorbate reductase activities indicative of a functional glutaredoxin. The protein is expressed at all stages of the T4 infection cycle and can serve as a hydrogen donor for the phage ribonucleotide reductase in in vitro experiments.

Glutaredoxin-3 from Escherichia coli: AMINO ACID SEQUENCE, 1H AND 15N NMR ASSIGNMENTS, AND STRUCTURAL ANALYSIS

The primary and secondary structure of glutaredoxin-3 (Grx3), a glutathione-disulfide oxidoreductase from Escherichia coli, has been determined. The amino acid sequence of Grx3 consists of 82 residues and contains a redox-active motif, Cys-Pro-Tyr-Cys, typical of the glutaredoxin family. Sequence comparison reveals a homology (33% identity) to that of glutaredoxin-1 (Grx1) from E. coli as well as to other members of the thioredoxin superfamily. In addition to the active site cysteine residues, Grx3 contains one additional cysteine (Cys65) corresponding to one of the two non-active site (or structural) cysteine residues present in mammalian glutaredoxins. The sequence-specific 1H and 15N nuclear magnetic resonance assignments of reduced Grx3 have been obtained. From a combined analysis of chemical shifts, 3JHNalpha coupling constants, sequential and medium range NOEs, and amide proton exchange rates, the secondary structure of reduced Grx3 was determined and found to be very similar to that inferred from amino acid sequence comparison to homologous proteins. The consequences of the proposed structural similarity to Grx1 are that Grx3, while possessing a largely intact GSH binding cleft, would have a very different spatial distribution of charged residues, most notably surrounding the active site cysteine residues and occurring in the proposed hydrophobic protein-protein interaction area. These differences may contribute to the observed very low Kcat of Grx3 as a reductant of insulin disulfides or as a hydrogen donor for ribonucleotide reductase. Thus, despite an identical active site disulfide motif and a similar secondary structure and tertiary fold, Grx3 and Grx1 display large functional differences in in vitro protein disulfide oxido-reduction reactions.

High-level expression of fully active human glutaredoxin (thioltransferase) in E. coli and characterization of Cys 7 to Ser mutant protein

Glutaredoxin (Grx) (12 kDa) is a hydrogen donor for ribonucleotide reductase and also a general GSH-disulfide reductase of importance for redox regulation. To overexpress human glutaredoxin in Escherichia coli, a cDNA encoding human Grx was modified and cloned into the vector pET-3d and expressed in E. coli BL21(DE3) by IPTG induction. High-level expression of Grx was verified by GSH-disulfide oxidoreductase activity, SDS-PAGE and immunoblotting analysis. The recombinant human Grx in its reduced form was purified to homogenity with 50% yield and exhibited the same dehydroascorbate reductase and hydrogen donor activity for ribonucleotide reductase ( K m ∼ 0.2 μM) as the human placenta protein. Human Grx contains a total of 5 half-cystine residues including a non-conserved Cys 7 residue and is easily oxidized to form dimers during storage. A Grx mutant Cys 7 to Ser was generated by site-directed mutagenesis and the protein was purified to homogeneity. The mutant protein showed full activity and exhibited a much reduced tendency to form dimers compared with the wild type protein. Peptide sequencing confirmed the mutation and removal of the N-terminal Met residue in both wild type and mutant proteins. Fluorescence spectra demonstrated only tyrosine fluorescence in human Grx with a peak at 310 nm which increased 20% upon reduction and decreased by addition of GSSG demonstrating that glutathionecontaining disulfides are excellent substrates.

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.

29] Glutaredoxin

This chapter focuses on glutaredoxin (Grx). Glutaredoxin (Grx) was discovered as a glutathione (GSH)-dependent hydrogen donor for ribonucleotide reductase (RR) in a mutant of Escherichia coli lacking thioredoxin (Trx), the classic dithiol hydrogen donor. Purification and characterization of E. coli Grx showed a small protein, which also exhibited a general GSH-disulfide oxidoreductase activity with clear similarities to previously described eukaryotic GSH-disulfide oxidoreductases (or transhydrogenases), later renamed thioltransferases. The thioltransferase has no relation to the GSH transferases, which were later discovered, and the reactions catalyzed are oxidoreductions rather than transferase reactions. The first mammalian Grx was isolated from calf thymus as a hydrogen donor for the corresponding ribonucleotide reductase. Determination of the amino acid sequence demonstrated the same active site, -Cys-Pro-Tyr-Cys-, and a protein with elongations at both the N- and C-terminal ends. Glutaredoxin has a GSH binding site, defined by NMR and site-directed mutagenesis. This GSH binding site is the rationale behind the activity of Grx to reduce glutathionyl mixed disulfides. This activity has been shown to be dependent on only the presence of the N-terminal of the active site half-cystines.

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.

Null thioredoxin and glutaredoxin Escherichia coli K-12 mutants have no enhanced sensitivity to mutagens due to a new GSH-dependent hydrogen donor and high increases in ribonucleotide reductase activity

This work investigates whether a mutator phenotype is associated to the simultaneous deficiency in thioredoxin and glutaredoxin, the two known hydrogen donors of ribonucleotide reductase. To this end, new Escherichia coli K-12 strains carrying delta trxA and/or grx::kan null mutations were constructed to monitor mutagenesis by selecting forward mutations to L-arabinose resistance. Highly sensitive and specific enzyme-linked immunoassays were developed to confirm that trx-grx- cells lacked thioredoxin and glutaredoxin. A number of remarkable properties were observed in the newly constructed thioredoxin- and glutaredoxin-deficient bacteria compared with the wild type cells. Thus, they (i) grew on minimal medium plates, suggesting that the presence of thioredoxin and glutaredoxin may not be absolutely essential for sulfate reduction; (ii) showed normal mutagenic sensitivities toward a wide variety of DNA-damaging agents, as compared with wild type cells and trx- or grx- single mutants; (iii) displayed 14% of GSH-dependent and 30% of NADPH-dependent ribonucleotide reduction capacity with CDP as substrate in the presence or the absence of exogenous ribonucleotide reductase, respectively; and (iv) showed very high levels of ribonucleotide reductase activity, which was increased from 19- to 23-fold. The existence of a new glutathione-dependent hydrogen donor for ribonucleotide reductase and the high activity levels of this enzyme in trx-grx- defective cells could explain that thioredoxin and the first discovered glutaredoxin are not essential for deoxyribonucleotide synthesis, even under mutagenic stress.

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.

Nuclear magnetic resonance studies of recombinant Escherichia coli glutaredoxin

Escherichia coli glutaredoxin (85 amino acid residues, Mr = 9100), the glutathione-dependent hydrogen donor for ribonucleotide reductase, was purified from an inducible lambda PL, expression system both with a natural isotope content and with uniform 15N labelling. This material was used for obtaining sequence-specific 1H magnetic resonance assignments and the identification of regular secondary structures in the oxidized form of the protein, which contains the redox-active disulfide Cys11-Pro-Tyr-Cys14. Oxidized glutaredoxin contains a four-stranded beta-sheet, with the peripheral strand 32-37 arranged parallel to the strand 2-7, which further combines with the two additional strands 61-64 and 67-69 in an antiparallel fashion. The protein further contains three helices extending approximately from residues 13-28, 45-54 and 72-84.