Fredrik Åslund

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 otherin 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.

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.

Redox Potentials of Glutaredoxins and Other Thiol-Disulfide Oxidoreductases of the Thioredoxin Superfamily Determined by Direct Protein-Protein Redox Equilibria

Glutaredoxins belong to the thioredoxin superfamily of structurally similar thiol-disulfide oxidoreductases catalyzing thiol-disulfide exchange reactions via reversible oxidation of two active-site cysteine residues separated by two amino acids (CX 1 X 2C). Standard state redox potential (E°′) values for glutaredoxins are presently unknown, and use of glutathione/glutathione disulfide (GSH/GSSG) redox buffers for determining E°′ resulted in variable levels of GSH-mixed disulfides. To overcome this complication, we have used reverse-phase high performance liquid chromatography to separate and quantify the oxidized and reduced forms present in the thiol-disulfide exchange reaction at equilibrium after mixing one oxidized and one reduced protein. This allowed for direct and quantitative pair-wise comparisons of the reducing capacities of the proteins and mutant forms. Equilibrium constants from pair-wise reaction with thioredoxin or its P34H mutant, which have accurately determined E°′ values from their redox equilibrium with NADPH catalyzed by thioredoxin reductase, allowed for transformation into standard state values. Using this new procedure, the standard state redox potentials for the Escherichia coliglutaredoxins 1 and 3, which contain identical active site sequences CPYC, were found to be E°′ = −233 and −198 mV, respectively. These values were confirmed independently by using the thermodynamic linkage between the stability of the disulfide bond and the stability of the protein to denaturation. Comparison of calculatedE°′ values from a number of proteins ranging from −270 mV for E. coli Trx to −124 mV for DsbA obtained using this method with those determined using glutathione redox buffers provides independent confirmation of the standard state redox potential of glutathione as −240 mV. Determining redox potentials through direct protein-protein equilibria is of general interest as it overcomes errors in determining redox potentials calculated from large equilibrium constants with the strongly reducing NADPH or by accumulating mixed disulfides with GSH.

Bridge over Troubled Waters: Sensing Stress by Disulfide Bond Formation

are members of the thioredoxin superfamily and exert their action by a disulfide exchange reaction utilizing a Cys-X1-X2-Cys active site. Other members of the thioredoxin superfamily are responsible for the introduction and isomerization of disulfide bonds that are often presFredrik Åslund† and Jon Beckwith* Department of Microbiology and Molecular Genetics Harvard Medical School 200 Longwood Avenue ent in secreted proteins. All evidence points to a situaBoston, Massachusetts 02115 tion in which cytosolic proteins have evolved to maintain their cysteines reduced in the native form, whereas many secreted proteins have evolved to be more stable The regulation of protein activity is a major factor in when their cysteines are joined in disulfide bonds. Thus, the cellular response to a changing environment. Wellchanges in the reducing environment of the cytosol can established mechanisms for such regulation include have profound effects on protein folding and activity. protein–protein interactions, allosteric changes generPerturbations of the cellular redox conditions can be ated by ligand binding, and chemical modifications such achieved either by mutations that eliminate components as phosphorylation. It has long been postulated that of the thioredoxin and glutaredoxin systems or by envidisulfide bond formation represents a covalent modifironmental oxidative stress. Mutant analysis shows that cation that can regulate protein activity. This proposal a simultaneous block of both disulfide-reducing pathhas come from studies in which enzymes or transcripways is incompatible with growth under aerobic condition factors were shown to lose activity after oxidation tions (Prinz et al., 1997). Functional overlap between the of cysteine residues in vitro, but to regain activity when two pathways is indicated by the finding that, in the exposed to the disulfide reductant thioredoxin. Howabsence of only one of the pathways, strains can survive ever, such reports may often be based on the fact that and grow reasonably well. Nevertheless, the oxidizing the proteins studied usually exist in the reducing enviconditions in the cytosol of such strains allows for the ronment of the cytosol; the oxidative inactivation obformation of disulfide bonds in some proteins. This disulserved may simply reflect the unnatural oxidizing condifide bond–forming activity can be monitored by expresstions of the in vitro system and not reflect the in vivo ing in the cytosol a normally exported protein, such as state of affairs. alkaline phosphatase, which requires disulfide bonds for Thus, while it is not clear that loss of function by its enzymatic activity. Although it has yet to be directly disulfide bond formation has been established as a regudemonstrated, it seems likely that unwanted disulfide latory mechanism, recent results suggest that the rebonds are also generated in the normal resident proteins verse process, gain of function by disulfide bond formaof the cytosol during oxidative stress—a situation we tion, may be a common way of responding to cellular refer to as “disulfide stress.” stress. The utilization of improved techniques for asBacteria encounter oxidative stresses in environsessing the disulfide-bonded states of proteins in vivo ments with high levels of hydrogen peroxide or other has allowed a reexamination of the role of these bonds in reactive oxygen species. One example of such stress regulating protein activity. These studies provide strong occurs when pathogenic bacteria confront oxidative evidence that two bacterial proteins, the transcription bursts upon invasion of eukaryotic host cells. These factor OxyR and the chaperone heat shock protein 33 encounters, in addition to damaging other cellular mole(Hsp33), are activated by the oxidation of cysteine resicules, may also cause the introduction of deleterious dues to disulfide bonds (Zheng et al., 1998; Jakob et al., disulfide bonds into proteins. Furthermore, studies us1999). These findings and the approaches used should ing cytosolic alkaline phosphatase suggest that E. coli provide impetus to a search for what is likely to be in the stationary phase is subject to disulfide stress a more widespread occurrence of this mechanism for (Dukan and Nyström, 1998). regulating protein activity. How does the bacterial cell respond to the stress The two most important means of maintaining the that disulfide bond formation in the cytosol poses? Two reducing thiol-disulfide status of the cytosol involve the features common to this response are the restoration thioredoxin-thioredoxin reductase pathway and the gluof the redox homeostasis in the cytosol and the eliminatathione-glutaredoxin pathway (Prinz et al., 1997). Thiotion of the harmful oxidant (Figure 1). Such responses redoxins and glutaredoxins were first detected by their are a requirement for life in an aerobic environment that ability to reduce a disulfide bond in the active site of is accompanied by exposure to reactive oxygen species. ribonucleotide reductase, as part of the reduction pathIn E. coli, the exposure to reactive oxygen species way converting ribonucleotides to deoxyribonucleo(ROS) such as O2· and hydrogen peroxide activates the tides. The reduced form of thioredoxin is regenerated transcription factors SoxR/S and OxyR (Hidalgo et al., by thioredoxin reductase, whereas glutaredoxin is kept 1997; Zheng et al., 1998). These factors trigger the exreduced by glutathione. Thioredoxin and glutaredoxin pression of defense activities including superoxide dismutase and peroxidases. Recently, it was discovered * To whom correspondence should be addressed (e-mail: jbeck that the response to peroxides and disulfide stress is with@hms.harvard.edu). due to the formation of a disulfide bond within the OxyR † Present address: Department of Biology, Massachusetts Institute protein, thus converting it to a transcriptional activator of Technology, Cambridge, Massachusetts 02139 (as of April 1,

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.

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 aV max 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 uponK m  PAPS (22.5 μm). Grx1 effectively replaced Trx1 with essentially identicalK m -values: K m  trx1(13.7 μm), K m  grx1 (14.9 μm), whereas the K m  trx2was considerably higher (34.2 μm). The results agree with previous in vivo data suggesting that Trx1 or Grx1 is essential for sulfate reduction but not for ribonucleotide reduction inE. coli.

The Ribonucleotide Reductase System of Lactococcus lactis CHARACTERIZATION OF AN NrdEF ENZYME AND A NEW ELECTRON TRANSPORT PROTEIN

Escherichia coli contains the genetic information for three separate ribonucleotide reductases. Two of them (class I enzymes), coded by the nrdAB and nrdEF genes, respectively, contain a tyrosyl radical, whose generation requires oxygen. The NrdAB enzyme is physiologically active. The function of the nrdEF gene is not known. The third enzyme (class III), coded by nrdDG, operates during anaerobiosis. The DNA of Lactococcus lactis contains sequences homologous to the nrdDG genes. Surprisingly, an nrdD mutant of L. lactis grew well under standard anaerobic growth conditions. The ribonucleotide reductase system of this mutant was shown to consist of an enzyme of the NrdEF-type and a small electron transport protein. The coding operon contains the nrdEF genes and two open reading frames, one of which (nrdH) codes for the small protein. The same gene organization is present in E. coli. We propose that the aerobic class I ribonucleotide reductases contain two subclasses, one coded by nrdAB, active in E. coli and eukaryotes (class Ia), the other coded by nrdEF, present in various microorganisms (class Ib). The NrdEF enzymes use NrdH proteins as electron transporter in place of thioredoxin or glutaredoxin used by NrdAB enzymes. The two classes also differ in their allosteric regulation by dATP.

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 β-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 (βαβαββα) 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.

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-Xaa1-Xaa2-Cys motif. The individual members of the family differ strongly in their intrinsic redox potentials. However, the role of the different redox potentials for thein 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 vivocharacterization 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.

Thioredoxin 1 Promotes Intracellular Replication and Virulence of Salmonella enterica Serovar Typhimurium

ABSTRACT The effect of the cytoplasmic reductase and protein chaperone thioredoxin 1 on the virulence of Salmonella enterica serovar Typhimurium was evaluated by deleting the trxA, trxB, or trxC gene of the cellular thioredoxin system, the grxA or gshA gene of the glutathione/glutaredoxin system, or the dsbC gene coding for a thioredoxin-dependent periplasmic disulfide bond isomerase. Mutants were tested for tolerance to oxidative and nitric oxide donor substances in vitro, for invasion and intracellular replication in cultured epithelial and macrophage-like cells, and for virulence in BALB/c mice. In these experiments only the gshA mutant, which was defective in glutathione synthesis, exhibited sensitization to oxidative stress in vitro and a small decrease in virulence. In contrast, the trxA mutant did not exhibit any growth defects or decreased tolerance to oxidative or nitric oxide stress in vitro, yet there were pronounced decreases in intracellular replication and mouse virulence. Complementation analyses using defined catalytic variants of thioredoxin 1 showed that there is a direct correlation between the redox potential of thioredoxin 1 and restoration of intracellular replication of the trxA mutant. Attenuation of mouse virulence that was caused by a deficiency in thioredoxin 1 was restored by expression of wild-type thioredoxin 1 in trans but not by expression of a catalytically inactive variant. These results clearly imply that in S. enterica serovar Typhimurium, the redox-active protein thioredoxin 1 promotes virulence, whereas in vitro tolerance to oxidative stress depends on production of glutathione.