A flavoprotein oxidase defines a new endoplasmic reticulum pathway for biosynthetic disulphide bond formation.

Ero1 and Pdi1 are essential elements of the pathway for the formation of disulphide bonds within the endoplasmic reticulum (ER). By screening for alternative oxidation pathways in Saccharomyces cerevisiae, we identified ERV2 as a gene that when overexpressed can restore viability and disulphide bond formation to an ero1-1 mutant strain. ERV2 encodes a luminal ER protein of relative molecular mass 22,000. Purified recombinant Erv2p is a flavoenzyme that can catalyse O2-dependent formation of disulphide bonds. Erv2p transfers oxidizing equivalents to Pdi1p by a dithiol-disulphide exchange reaction, indicating that the Erv2p-dependent pathway for disulphide bond formation closely parallels that of the previously identified Ero1p-dependent pathway.

Structural basis for the thioredoxin-like activity profile of the glutaredoxin-like NrdH-redoxin from Escherichia coli.

NrdH-redoxin is a representative of a class of small redox proteins that contain a conserved CXXC motif and are characterized by a glutaredoxin-like amino acid sequence and thioredoxin-like activity profile. The crystal structure of recombinant Escherichia coli NrdH-redoxin in the oxidized state has been determined at 1.7 A resolution by multiwavelength anomalous diffraction. NrdH-redoxin belongs to the thioredoxin superfamily and is structurally most similar to E. coli glutaredoxin 3 and phage T4 glutaredoxin. The angle between the C-terminal helix alpha3 and strand beta4, which differs between thioredoxin and glutaredoxin, has an intermediate value in NrdH-redoxin. The orientation of this helix is to a large extent determined by an extended hydrogen-bond network involving the highly conserved sequence motif (61)WSGFRP(D/E)(67), which is unique to this subclass of the thioredoxin superfamily. Residues that bind glutathione in glutaredoxins are in general not conserved in NrdH-redoxin, and no glutathione-binding cleft is present. Instead, NrdH-redoxin contains a wide hydrophobic pocket at the surface, similar to thioredoxin. Modeling studies suggest that NrdH-redoxin can interact with E. coli thioredoxin reductase at this pocket and also via a loop that is complementary to a crevice in the reductase in a similar manner as observed in the E. coli thioredoxin-thioredoxin reductase complex.

NMR structure of oxidized glutaredoxin 3 from Escherichia coli.

A high precision NMR structure of oxidized glutaredoxin 3 [C65Y] from Escherichia coli has been determined. The conformation of the active site including the disulphide bridge is highly similar to those in glutaredoxins from pig liver and T4 phage. A comparison with the previously determined structure of glutaredoxin 3 [C14S, C65Y] in a complex with glutathione reveals conformational changes between the free and substrate-bound form which includes the sidechain of the conserved, active site tyrosine residue. In the oxidized form this tyrosine is solvent exposed, while it adopts a less exposed conformation, stabilized by hydrogen bonds, in the mixed disulfide with glutathione. The structures further suggest that the formation of a covalent linkage between glutathione and glutaredoxin 3 is necessary in order to induce these structural changes upon binding of the glutathione peptide. This could explain the observed low affinity of glutaredoxins for S-blocked glutathione analogues, in spite of the fact that glutaredoxins are highly specific reductants of glutathione mixed disulfides.

Thioredoxin 2 is involved in the oxidative stress response in Escherichia coli.

Two genes encoding thioredoxin are found on the Escherichia coli genome. Both of them are capable of reducing protein disulfide bonds in vivo and in vitro. The catalytic site contains a Cys-X(1)-X(2)-Cys motif in a so-called thioredoxin fold. Thioredoxin 2 has two additional pairs of cysteines in a non-conserved N-terminal domain. This domain does not appear to be important for the function of thioredoxin 2 in donating electrons to ribonucleotide reductase, 3'-phosphoadenylsulfate-reductase, or the periplasmic disulfide isomerase DsbC. Our results suggests that the two thioredoxins are equivalent for most of the in vivo functions that were tested. On the other hand, transcriptional regulation is different. The expression of trxC is regulated by the transcriptional activator OxyR in response to oxidative stress. Oxidized OxyR binds directly to the trxC promoter and induces its expression in response to elevated hydrogen peroxide levels or the disruption of one or several of the cytoplasmic redox pathways. Mutants lacking thioredoxins 1 and 2 are more resistant to high levels of hydrogen peroxide, whereas they are more sensitive to diamide, a disulfide bond-inducing agent.

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.

Efficient folding of proteins with multiple disulfide bonds in the Escherichia coli cytoplasm.

Under physiological conditions, the Escherichia coli cytoplasm is maintained in a reduced state that strongly disfavors the formation of stable disulfide bonds in proteins. However, mutants in which the reduction of both thioredoxins and glutathione is impaired (trxB gor mutants) accumulate oxidized, enzymatically active alkaline phosphatase in the cytoplasm. These mutants grow very poorly in the absence of an exogenous reductant and accumulate extragenic suppressors at a high frequency. One such suppressor strain, FA113, grows almost as rapidly as the wild type in the absence of reductant, exhibits slightly faster kinetics of disulfide bond formation, and has fully induced activity of the transcriptional activator, OxyR. FA113 gave substantially higher yields of properly oxidized proteins compared with wild-type or trxB mutant strains. For polypeptides with very complex patterns of disulfide bonds, such as vtPA and the full-length tPA, the amount of active protein was further enhanced up to 15-fold by co-expression of TrxA (thioredoxin 1) mutants with different redox potentials, or 20-fold by the protein disulfide isomerase, DsbC. Remarkably, higher yields of oxidized, biologically active proteins were obtained by expression in the cytoplasm of E. coli FA113 compared with what could be achieved via secretion into the periplasm of a wild-type strain, even under optimized conditions. These results demonstrate that the cytoplasm can be rendered sufficiently oxidizing to allow efficient formation of native disulfide bonds without compromising cell viability.

Importance of redox potential for the in vivo function of the cytoplasmic disulfide reductant thioredoxin from Escherichia coli.

The thioredoxin superfamily consists of enzymes that catalyze the reduction, formation, and isomerization of disulfide bonds and exert their activity through a redox active disulfide in a Cys-Xaa(1)-Xaa(2)-Cys motif. The individual members of the family differ strongly in their intrinsic redox potentials. However, the role of the different redox potentials for the in vivo function of these enzymes is essentially unknown. To address the question of in vivo importance of redox potential for the most reducing member of the enzyme family, thioredoxin, we have employed a set of active site variants of thioredoxin with increased redox potentials (-270 to -195 mV) for functional studies in the cytoplasm of Escherichia coli. The variants proved to be efficient substrates of thioredoxin reductase, providing a basis for an in vivo characterization of NADPH-dependent reductive processes catalyzed by the thioredoxin variants. The reduction of sulfate and methionine sulfoxide, as well as the isomerization of periplasmic disulfide bonds by DsbC, which all depend on thioredoxin as catalyst in the E. coli cytoplasm, proved to correlate well with the intrinsic redox potentials of the variants in complementation assays. The same correlation could be established in vitro by using the thioredoxin-catalyzed reduction of lipoic acid by NADPH as a model reaction. We propose that the rate of direct reduction of substrates by thioredoxin, which largely depends on the redox potential of thioredoxin, is the most important parameter for the in vivo function of thioredoxin, as recycling of reduced thioredoxin through NADPH and thioredoxin reductase is not rate-limiting for its catalytic cycle.

Direct NMR observation of the Cys-14 thiol proton of reduced Escherichia coli glutaredoxin-3 supports the presence of an active site thiol-thiolate hydrogen bond.

The active site of Escherichia coli glutaredoxin-3 (Grx3) consists of two redox active cysteine residues in the sequence -C11-P-Y-C14-H-. The 1H NMR resonance of the cysteine thiol proton of Cys-14 in reduced Grx3 is observed at 7.6 ppm. The large downfield shift and NOEs observed with this thiol proton resonance suggest the presence of a hydrogen bond with the Cys-11 thiolate, which is shown to have an abnormally low pKa value. A hydrogen bond would also agree with activity data of Grx3 active site mutants. Furthermore, the activity is reduced in a Grx3 H15V mutant, indicating electrostatic contributions to the stabilization of the Cys-11 thiolate.

Regulation of the OxyR transcription factor by hydrogen peroxide and the cellular thiol-disulfide status.

The Escherichia coli transcription factor OxyR is activated by the formation of an intramolecular disulfide bond and subsequently is deactivated by enzymatic reduction of the disulfide bond. Here we show that OxyR can be activated by two possible pathways. In mutants defective in the cellular disulfide-reducing systems, OxyR is constitutively activated by a change in the thiol-disulfide redox status in the absence of added oxidants. In wild-type cells, OxyR is activated by hydrogen peroxide. By monitoring the presence of the OxyR disulfide bond after exposure to hydrogen peroxide in vivo and in vitro, we also show that the kinetics of OxyR oxidation by low concentrations of hydrogen peroxide is significantly faster than the kinetics of OxyR reduction, allowing for transient activation in an overall reducing environment. We propose that the activity of OxyR in vivo is determined by the balance between hydrogen peroxide levels and the cellular redox environment.

New thioredoxins and glutaredoxins as electron donors of 3'-phosphoadenylylsulfate reductase.

Reduction of inorganic sulfate to sulfite in prototrophic bacteria occurs with 3'-phosphoadenylylsulfate (PAPS) as substrate for PAPS reductase and is the first step leading to reduced sulfur for cellular biosynthetic reactions. The relative efficiency as reductants of homogeneous highly active PAPS reductase of the newly identified second thioredoxin (Trx2) and glutaredoxins (Grx1, Grx2, Grx3, and a mutant Grx1C14S) was compared with the well known thioredoxin (Trx1) from Escherichia coli. Trx1, Trx2, and Grx1 supported virtually identical rates of sulfite formation with a Vmax ranging from 6.6 units mg-1 (Trx1) to 5.1 units mg-1 (Grx1), whereas Grx1C14S was only marginally active, and Grx2 and Grx3 had no activity. The structural difference between active reductants had no effect upon Km PAPS (22.5 microM). Grx1 effectively replaced Trx1 with essentially identical Km-values: Km trx1 (13.7 microM), Km grx1 (14.9 microM), whereas the Km trx2 was considerably higher (34.2 microM). The results agree with previous in vivo data suggesting that Trx1 or Grx1 is essential for sulfate reduction but not for ribonucleotide reduction in E. coli.

Reactivity of glutaredoxins 1, 2 and 3 from Escherichia coli and protein disulfide isomerase towards glutathionyl-mixed disulfides in ribonuclease A.

We have examined the activity of protein disulfide isomerase (PDI) and glutaredoxin (Grx) 1, 2 and 3 from Escherichia coli to catalyze the cleavage of glutathionylated ribonuclease A (RNase-SG) by 1 mM GSH to yield reduced RNase. Apparent Km values for RNase-SG were similar, 2-10 microM, for Grx 1, 3 and PDI but Grx I and Grx 3 showed 500-fold higher turnover numbers than PDI. The atypical Grx 2 also catalyzed deglutathionylation by GSH, but had higher Km and apparent turnover number values compared to the two classical Grx. Refolding of RNase in a glutathione redox buffer was catalyzed by PDI. However, it could be measured only after a characteristic lag phase that was shortened by all three E. coli Grxs in a concentration-dependent manner. A role of the glutaredoxin mechanism in the endoplasmic reticulum is suggested.

Disulfide bond formation in the Escherichia coli cytoplasm: an in vivo role reversal for the thioredoxins.

Cytoplasmic proteins do not generally contain structural disulfide bonds, although certain cytoplasmic enzymes form such bonds as part of their catalytic cycles. The disulfide bonds in these latter enzymes are reduced in Escherichia coli by two systems; the thioredoxin pathway and the glutathione/glutaredoxin pathway. However, structural disulfide bonds can form in proteins in the cytoplasm when the gene (trxB) for the enzyme thioredoxin reductase is inactivated by mutation. This disulfide bond formation can be detected by assessing the state of the normally periplasmic enzyme alkaline phosphatase (AP) when it is localized to the cytoplasm. Here we show that the formation of disulfide bonds in cytoplasmic AP in the trxB mutant is dependent on the presence of two thioredoxins in the cell, thioredoxins 1 and 2, the products of the genes trxA and trxC, respectively. Our evidence supports a model in which the oxidized forms of these thioredoxins directly catalyze disulfide bond formation in cytoplasmic AP, a reversal of their normal role. In addition, we show that the recently discovered thioredoxin 2 can perform many of the roles of thioredoxin 1 in vivo, and thus is able to reduce certain essential cytoplasmic enzymes. Our results suggest that the three most effective cytoplasmic disulfide-reducing proteins are thioredoxin 1, thioredoxin 2 and glutaredoxin 1; expression of any one of these is sufficient to support aerobic growth. Our results help to explain how the reducing environment in the cytoplasm is maintained so that disulfide bonds do not normally occur.

Glutaredoxin function for the carboxyl-terminal domain of the plant-type 5'-adenylylsulfate reductase.

5'-Adenylylsulfate (APS) reductase (EC 1.8.99.-) catalyzes the reduction of activated sulfate to sulfite in plants. The evidence presented here shows that a domain of the enzyme is a glutathione (GSH)-dependent reductase that functions similarly to the redox cofactor glutaredoxin. The APR1 cDNA encoding APS reductase from Arabidopsis thaliana is able to complement the cysteine auxotrophy of an Escherichia coli cysH [3'-phosphoadenosine-5'-phosphosulfate (PAPS) reductase] mutant, only if the E. coli strain produces glutathione. The purified recombinant enzyme (APR1p) can use GSH efficiently as a hydrogen donor in vitro, showing aKm[GSH] approximately of 0.6 mM. Gene dissection was used to express separately the regions of APR1p from amino acids 73-327 (the R domain), homologous with microbial PAPS reductase, and from amino acids 328-465 (the C domain), homologous with thioredoxin. The R and C domains alone are inactive in APS reduction, but the activity is partially restored by mixing the two domains. The C domain shows a number of activities that are typical of E. coli glutaredoxin rather than thioredoxin. Both the C domain and APR1p are highly active in GSH-dependent reduction of hydroxyethyldisulfide, cystine, and dehydroascorbate, showing a Km[GSH] in these assays of approximately 1 mM. The R domain does not show these activities. The C domain is active in GSH-dependent reduction of insulin disulfides and ribonucleotide reductase, whereas APR1p and R domain are inactive. The C domain can substitute for glutaredoxin in vivo as demonstrated by complementation of an E. coli mutant, underscoring the functional similarity between the two enzymes.

Rat and calf thioredoxin reductase are homologous to glutathione reductase with a carboxyl-terminal elongation containing a conserved catalytically active penultimate selenocysteine residue.

We have determined the sequence of 23 peptides from bovine thioredoxin reductase covering 364 amino acid residues. The result was used to identify a rat cDNA clone (2.19 kilobase pairs), which contained an open reading frame of 1496 base pairs encoding a protein with 498 residues. The bovine and rat thioredoxin reductase sequences revealed a close homology to glutathione reductase including the conserved active site sequence (Cys-Val-Asn-Val-Gly-Cys). This also confirmed the identity of a previously published putative human thioredoxin reductase cDNA clone. Moreover, one peptide of the bovine enzyme contained a selenocysteine residue in the motif Gly-Cys-SeCys-Gly (where SeCys represents selenocysteine). This motif was conserved at the carboxyl terminus of the rat and human enzymes, provided that TGA in the sequence GGC TGC TGA GGT TAA, being identical in both cDNA clones, is translated as selenocysteine and that TAA confers termination of translation. The 3'-untranslated region of both cDNA clones contained a selenocysteine insertion sequence that may form potential stem loop structures typical of eukaryotic selenocysteine insertion sequence elements required for the decoding of UGA as selenocysteine. Carboxypeptidase Y treatment of bovine thioredoxin reductase after reduction by NADPH released selenocysteine from the enzyme with a concomitant loss of enzyme activity measured as reduction of thioredoxin or 5,5'-dithiobis(2-nitrobenzoic acid). This showed that the carboxyl-terminal motif was essential for the catalytic activity of the enzyme.

Activation of the OxyR transcription factor by reversible disulfide bond formation.

The OxyR transcription factor is sensitive to oxidation and activates the expression of antioxidant genes in response to hydrogen peroxide in Escherichia coli. Genetic and biochemical studies revealed that OxyR is activated through the formation of a disulfide bond and is deactivated by enzymatic reduction with glutaredoxin 1 (Grx1). The gene encoding Grx1 is regulated by OxyR, thus providing a mechanism for autoregulation. The redox potential of OxyR was determined to be -185 millivolts, ensuring that OxyR is reduced in the absence of stress. These results represent an example of redox signaling through disulfide bond formation and reduction.

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.

The role of the thioredoxin and glutaredoxin pathways in reducing protein disulfide bonds in the Escherichia coli cytoplasm.

In Escherichia coli, two pathways use NADPH to reduce disulfide bonds that form in some cytoplasmic enzymes during catalysis: the thioredoxin system, which consists of thioredoxin reductase and thioredoxin, and the glutaredoxin system, composed of glutathione reductase, glutathione, and three glutaredoxins. These systems may also reduce disulfide bonds which form spontaneously in cytoplasmic proteins when E. coli is grown aerobically. We have investigated the role of both systems in determining the thiol-disulfide balance in the cytoplasm by determining the ability of protein disulfide bonds to form in mutants missing components of these systems. We find that both the thioredoxin and glutaredoxin systems contribute to reducing disulfide bonds in cytoplasmic proteins. In addition, these systems can partially substitute for each other in vivo since double mutants missing parts of both systems generally allow substantially more disulfide bond formation than mutants missing components of just one system. Some of these double mutants were found to require the addition of a disulfide reductant to the medium to grow well aerobically. Thus, E. coli requires either a functional thioredoxin or glutaredoxin system to reduce disulfide bonds which appear after each catalytic cycle in the essential enzyme ribonucleotide reductase and perhaps to reduce non-native disulfide bonds in cytoplasmic proteins. Our results suggest the existence of a novel thioredoxin in E. coli.

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.