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.

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.

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 coexpression 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. The fundamental discovery that a denatured protein, ribonuclease, could assemble correctly in the absence of any catalysts indicated that all of the information for the proper folding of a protein was present in its primary amino acid sequence. Because disulfide bonds are necessary for the proper folding of ribonuclease, these experiments were also taken to mean that disulfide bond formation was independent of enzyme catalysts. Thus, it had been presumed that only the presence of oxygen (or small molecules such as oxidized glutathione) is needed in vivo for disulfide bond formation. This presumption appeared to explain the fact that proteins with structural disulfide bonds are only found in the more oxidizing noncytosolic intracellular compartments or in the extracellular space. According to this view, disulfide bonds do not form in the cytosol simply because the reducing components such as glutathione and thioredoxins keep such bonds reduced.The first modification of this view of disulfide bond formation and the basis for its compartmentalization came from the finding that disulfide bond formation in Gram-negative bacteria does require the presence of a protein catalyst, DsbA (1-5). This finding not only changed the picture of how disulfide bond formation takes place normally but also raised questions about the basis for the absence of disulfide bonds in cytosolic proteins. Normally, the formation of stable disulfide bonds in the cytoplasm is an exceedingly rar...

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.

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 2 C). 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 reversephase 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 pairwise 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 coli glutaredoxins 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 calculated E° 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 proteinprotein 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.Glutaredoxin (Grx1) 1 was discovered as a GSH-dependent hydrogen donor for ribonucleotide reductase in Escherichia coli mutants lacking the first identified electron donor, thioredoxin (1). The presence of an additional hydrogen donor system for ribonucleotide reductase was postulated since a double mutant lacking both Grx1 and Trx was viable (2). The search for this third hydrogen donor system resulted in the isolation of two additional glutaredoxins in E. coli, Grx2 and Grx3 (3). The recent structural characterization of Grx3 showed that in addition to the 33% amino acid sequence identity with Grx1, the two proteins have highly conserved secondary structure elements and overall fold (4). However, despite Grx1 and Grx3 being closely related 9-kDa redox proteins with identical active-site sequences (CPYC), Grx3 exhibits only a fraction of the activity of Grx1 as a reductant of ribonucleotide reductase (NrdAB and NrdEF; Refs. 3 and 5) or of insulin disulfides (4). ...

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.

Staphylococcus aureus is the leading cause of wound and hospitalacquired infections worldwide. The emergence of S. aureus strains with resistance to multiple antibiotics requires the identification of bacterial virulence genes and the development of novel therapeutic strategies. Herein, bursa aurealis, a mariner-based transposon, was used for random mutagenesis and for the isolation of 10,325 S. aureus variants with defined insertion sites. By screening for loss-of-function mutants in a Caenorhabditis elegans killing assay, 71 S. aureus virulence genes were identified. Some of these genes are also required for S. aureus abscess formation in a murine infection model. transposon ͉ Caenorhabditis elegans

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.Thioredoxin reductase is a dimeric enzyme with a redoxactive disulfide and an FAD in each monomer, and it is a member of a larger family of pyridine nucleotide-disulfide oxidoreductases, which includes the closely related enzymes lipoamide dehydrogenase, glutathione reductase, trypanothione reductase, and mercuric ion reductase (1). Thioredoxin reductase (TrxR) 1 catalyzes the NADPH-dependent reduction of the active site disulfide in oxidized thioredoxin (Trx-S 2 ) to give a dithiol in reduced thioredoxin (Trx-(SH) 2 ).