Reactivity of thioredoxin as a protein thiol-disulfide oxidoreductase.
Ontology highlight
ABSTRACT:
SUBMITTER: Cheng Z
PROVIDER: S-EPMC3212873 | biostudies-literature | 2011 Sep
REPOSITORIES: biostudies-literature
Publications
Reactivity of thioredoxin as a protein thiol-disulfide oxidoreductase.
Cheng Zhiyong Z Zhang Jinfeng J Ballou David P DP Williams Charles H CH
Chemical reviews 20110727 9
Similar Datasets
Project description:The thiol-disulfide oxidoreductase CXXC catalytic domain of thioredoxin contributes to antioxidant defense in phylogenetically diverse organisms. We find that although the oxidoreductase activity of thioredoxin-1 protects Salmonella enterica serovar Typhimurium from hydrogen peroxide in vitro, it does not appear to contribute to Salmonella's antioxidant defenses in vivo. Nonetheless, thioredoxin-1 defends Salmonella from oxidative stress resulting from NADPH phagocyte oxidase macrophage expression during the innate immune response in mice. Thioredoxin-1 binds to the flexible linker, which connects the receiver and effector domains of SsrB, thereby keeping this response regulator in the soluble fraction. Thioredoxin-1, independently of thiol-disulfide exchange, activates intracellular SPI2 gene transcription required for Salmonella resistance to both reactive species generated by NADPH phagocyte oxidase and oxygen-independent lysosomal host defenses. These findings suggest that the horizontally acquired virulence determinant SsrB is regulated post-translationally by ancestrally present thioredoxin.
| S-EPMC4930247 | biostudies-literature
Project description:Cytoplasmic desulfothioredoxin (Dtrx) from the anaerobe Desulfovibrio vulgaris Hildenborough has been identified as a new member of the thiol disulfide oxidoreductase family. The active site of Dtrx contains a particular consensus sequence, CPHC, never seen in the cytoplasmic thioredoxins and generally found in periplasmic oxidases. Unlike canonical thioredoxins (Trx), Dtrx does not present any disulfide reductase activity, but it presents instead an unusual disulfide isomerase activity. We have used NMR spectroscopy to gain insights into the structure and the catalytic mechanism of this unusual Dtrx. The redox potential of Dtrx (-181 mV) is significantly less reducing than that of canonical Trx. A pH dependence study allowed the determination of the pK(a) of all protonable residues, including the cysteine and histidine residues. Thus, the pK(a) values for the thiol group of Cys(31) and Cys(34) are 4.8 and 11.3, respectively. The His(33) pK(a) value, experimentally determined for the first time, differs notably as a function of the redox states, 7.2 for the reduced state and 4.6 for the oxidized state. These data suggest an important role for His(33) in the molecular mechanism of Dtrx catalysis that is confirmed by the properties of mutant DtrxH33G protein. The NMR structure of Dtrx shows a different charge repartition compared with canonical Trx. The results presented are likely indicative of the involvement of this protein in the catalysis of substrates specific of the anaerobe cytoplasm of DvH. The study of Dtrx is an important step toward revealing the molecular details of the thiol-disulfide oxidoreductase catalytic mechanism.
| S-EPMC3265852 | biostudies-literature
Project description:The bacterial protein DsbD transfers reductant from the cytoplasm to the otherwise oxidizing environment of the periplasm. This reducing power is required for several essential pathways, including disulfide bond formation and cytochrome c maturation. DsbD includes a transmembrane domain (tmDsbD) flanked by two globular periplasmic domains (nDsbD/cDsbD); each contains a cysteine pair involved in electron transfer via a disulfide exchange cascade. The final step in the cascade involves reduction of the Cys(103)-Cys(109) disulfide of nDsbD by Cys(461) of cDsbD. Here we show that a complex between the globular periplasmic domains is trapped in vivo only when both are linked by tmDsbD. We have found previously ( Mavridou, D. A., Stevens, J. M., Ferguson, S. J., & Redfield, C. (2007) J. Mol. Biol. 370, 643-658 ) that the attacking cysteine (Cys(461)) in isolated cDsbD has a high pK(a) value (10.5) that makes this thiol relatively unreactive toward the target disulfide in nDsbD. Here we show using NMR that active-site pK(a) values change significantly when cDsbD forms a complex with nDsbD. This modulation of pK(a) values is critical for the specificity and function of cDsbD. Uncomplexed cDsbD is a poor nucleophile, allowing it to avoid nonspecific reoxidation; however, in complex with nDsbD, the nucleophilicity of cDsbD increases permitting reductant transfer. The observation of significant changes in active-site pK(a) values upon complex formation has wider implications for understanding reactivity in thiol:disulfide oxidoreductases.
| S-EPMC2631958 | biostudies-literature
Project description:Sulfate reducers have developed a multifaceted adaptative strategy to survive against oxidative stresses. Along with this oxidative stress response, we recently characterized an elegant reversible disulfide bond-dependent protective mechanism in the pyruvate:ferredoxin oxidoreductase (PFOR) of various Desulfovibrio species. Here, we searched for thiol redox systems involved in this mechanism. Using thiol fluorescent labeling, we show that glutathione is not the major thiol/disulfide balance-controlling compound in four different Desulfovibrio species and that no other plentiful low molecular weight thiol can be detected. Enzymatic analyses of two thioredoxins (Trxs) and three thioredoxin reductases allow us to propose the existence of two independent Trx systems in Desulfovibrio vulgaris Hildenborough (DvH). The TR1/Trx1 system corresponds to the typical bacterial Trx system. We measured a TR1 apparent K(m) value for Trx1 of 8.9 μM. Moreover, our results showed that activity of TR1 was NADPH-dependent. The second system named TR3/Trx3 corresponds to an unconventional Trx system as TR3 used preferentially NADH (K(m) for NADPH, 743 μM; K(m) for NADH, 5.6 μM), and Trx3 was unable to reduce insulin. The K(m) value of TR3 for Trx3 was 1.12 μM. In vitro experiments demonstrated that the TR1/Trx1 system was the only one able to reactivate the oxygen-protected form of Desulfovibrio africanus PFOR. Moreover, ex vivo pulldown assays using the mutant Trx1(C33S) as bait allowed us to capture PFOR from the DvH extract. Altogether, these data demonstrate that PFOR is a new target for Trx1, which is probably involved in the protective switch mechanism of the enzyme.
| S-EPMC3048668 | biostudies-literature
Project description:Disulfide bond (Dsb) oxidoreductases involved in oxidative protein folding govern bacterial survival and virulence. Over the past decade, oligomerization has emerged as a potential factor that dictates oxidoreductase activities. To investigate the role of oligomerization, we studied three Dsb-like ScsC oxidoreductases involved in copper resistance: the monomeric Salmonella enterica StScsC, and the trimeric Proteus mirabilis PmScsC and Caulobacter crescentus CcScsC. For copper sequestration, ScsC proteins must remain in the reduced form. However, all three ScsC proteins exhibit both dithiol oxidation and disulfide reduction activity, despite structural differences and previously reported limited in vitro activity. Most ScsC reductase activity relies on interactions with E. coli DsbD reductase, while oxidase activity depends on environmental oxidation. Interestingly, engineered monomeric PmScsC interacts effectively with the E. coli DsbB oxidase, at the partial expense of its reductase activity. These findings highlight oligomerization of oxidoreductases as a steric hindrance strategy to block undesirable upstream oxidative interactions.
| S-EPMC11634996 | biostudies-literature
Project description:We previously identified a novel thiol-disulfide oxidoreductase, SdbA, in Streptococcus gordonii that formed disulfide bonds in substrate proteins and played a role in multiple phenotypes. In this study, we used mutational, phenotypic, and biochemical approaches to identify and characterize the redox partners of SdbA. Unexpectedly, the results showed that SdbA has multiple redox partners, forming a complex oxidative protein-folding pathway. The primary redox partners of SdbA that maintain its active site in an oxidized state are a surface-exposed thioredoxin family lipoprotein called SdbB (Sgo_1171) and an integral membrane protein annotated as CcdA2. Inactivation of sdbB and ccdA2 simultaneously, but not individually, recapitulated the sdbA mutant phenotype. The sdbB-ccdA2 mutant had defects in a range of cellular processes, including autolysis, bacteriocin production, genetic competence, and extracellular DNA (eDNA) release. AtlS, the natural substrate of SdbA produced by the sdbB-ccdA2 mutant lacked activity and an intramolecular disulfide bond. The redox state of SdbA in the sdbB-ccdA2 mutant was found to be in a reduced form and was restored when sdbB and ccdA2 were knocked back into the mutant. In addition, we showed that SdbB formed a disulfide-linked complex with SdbA in the cell. Recombinant SdbB and CcdA2 exhibited oxidase activity and reoxidized reduced SdbA in vitro Collectively, our results demonstrate that S. gordonii uses multiple redox partners for oxidative protein folding.IMPORTANCEStreptococcus gordonii is a commensal bacterium of the human dental plaque. Previously, we identified an enzyme, SdbA, that forms disulfide bonds in substrate proteins and plays a role in a number of cellular processes in S. gordonii Here, we identified the redox partners of SdbA. We showed that SdbA has multiple redox partners, SdbB and CcdA2, forming a complex oxidative protein-folding pathway. This pathway is essential for autolysis, bacteriocin production, genetic competence, and extracellular DNA (eDNA) release in S. gordonii These cellular processes are considered to be important for the success of S. gordonii as a dental plaque organism. This is the first example of an oxidative protein-folding pathway in Gram-positive bacteria that consists of an enzyme that uses multiple redox partners to function.
| S-EPMC6482928 | biostudies-literature
Project description:Identification of targets of the protein disulfide reductase thioredoxin using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and thiol specific differential labeling with isotope-coded affinity tags (ICAT). Reduction of specific target disulfides is quantified by measuring ratios of cysteine residues labeled with the heavy (13C) and light (12C) ICAT reagents in peptides derived from tryptic digests of Trx-treated and non-treated samples. Keywords: protein, LC-MS/MS, ICAT
2008-11-20 | GSE11650 | GEO
Project description:Thiamine (vitamin B1), under the proper conditions, is able to reversibly open the thiazole ring, forming a thiol-bearing molecule that can be further oxidized to the corresponding disulfide. To improve the bioavailability of the vitamin, several derivatives of thiamine in the thioester or disulfide form were developed and extensively studied over time, as apparent from the literature. We have examined three thiamine-derived disulfides: thiamine disulfide, sulbutiamine, and fursultiamine with reference to their intervention in modulating the thiol redox state. First, we observed that both glutathione and thioredoxin (Trx) systems were able to reduce the three disulfides. In particular, thioredoxin reductase (TrxR) reduced these disulfides either directly or in the presence of Trx. In Caco-2 cells, the thiamine disulfide derivatives did not modify the total thiol content, which, however, was significantly decreased by the concomitant inhibition of TrxR. When oxidative stress was induced by tert-butyl hydroperoxide, the thiamine disulfides exerted a protective effect, indicating that the thiol form deriving from the reduction of the disulfides might be the active species. Further, the thiamine disulfides examined were shown to increase the nuclear levels of the transcription factor nuclear factor erythroid 2 related factor 2 and to stimulate both expression and activity of NAD(P)H quinone dehydrogenase 1 and TrxR. However, other enzymes of the glutathione and Trx systems were scarcely affected. As the thiol redox balance plays a critical role in oxidative stress and inflammation, the information presented can be of interest for further research, considering the potential favorable effect exerted in the cell by many sulfur compounds, including the thiamine-derived disulfides.
| S-EPMC11681303 | biostudies-literature
Project description:The actinobacterium Corynebacterium matruchotii has been implicated in nucleation of oral microbial consortia leading to biofilm formation. Due to the lack of genetic tools, little is known about basic cellular processes, including protein secretion and folding, in this organism. We report here a survey of the C. matruchotii genome, which encodes a large number of exported proteins containing paired cysteine residues, and identified an oxidoreductase that is highly homologous to the Corynebacterium diphtheriae thiol-disulfide oxidoreductase MdbA (MdbACd). Crystallization studies uncovered that the 1.2-Å resolution structure of C. matruchotii MdbA (MdbACm) possesses two conserved features found in actinobacterial MdbA enzymes, a thioredoxin-like fold and an extended α-helical domain. By reconstituting the disulfide bond-forming machine in vitro, we demonstrated that MdbACm catalyzes disulfide bond formation within the actinobacterial pilin FimA. A new gene deletion method supported that mdbA is essential in C. matruchotii Remarkably, heterologous expression of MdbACm in the C. diphtheriae ΔmdbA mutant rescued its known defects in cell growth and morphology, toxin production, and pilus assembly, and this thiol-disulfide oxidoreductase activity required the catalytic motif CXXC. Altogether, the results suggest that MdbACm is a major thiol-disulfide oxidoreductase, which likely mediates posttranslocational protein folding in C. matruchotii by a mechanism that is conserved in ActinobacteriaIMPORTANCE The actinobacterium Corynebacterium matruchotii has been implicated in the development of oral biofilms or dental plaque; however, little is known about the basic cellular processes in this organism. We report here a high-resolution structure of a C. matruchotii oxidoreductase that is highly homologous to the Corynebacterium diphtheriae thiol-disulfide oxidoreductase MdbA. By biochemical analysis, we demonstrated that C. matruchotii MdbA catalyzes disulfide bond formation in vitro Furthermore, a new gene deletion method revealed that deletion of mdbA is lethal in C. matruchotii Remarkably, C. matruchotii MdbA can replace C. diphtheriae MdbA to maintain normal cell growth and morphology, toxin production, and pilus assembly. Overall, our studies support the hypothesis that C. matruchotii utilizes MdbA as a major oxidoreductase to catalyze oxidative protein folding.
| S-EPMC5892113 | biostudies-literature
Project description:Selenoprotein F (SelF) is an endoplasmic reticulum-residing eukaryotic protein that contains a selenocysteine (Sec) residue. It has been suggested to be involved in a number of physiological processes by acting as a thiol-disulfide oxidoreductase, but the exact role has remained unclear due to the lack of a reliable production method. We document herein a robust synthesis of the human SelF through a three-segment two-ligation semisynthesis strategy. Highlighted in this synthetic route are the use of a mild desulfurization process to protect the side-chain of the Sec residue from being affected and the simultaneous removal of acetamidomethyl and p-methoxybenzyl protection groups by PdCl2, thus facilitating the synthesis of multi-milligrams of homogenous SelF. The reduction potential of SelF was determined and the thiol-disulfide oxidoreductase activity was further supported by its ability to catalyze the reduction and isomerization of disulfide bonds.
| S-EPMC9159075 | biostudies-literature