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The Gram-positive pathogen Corynebacterium diphtheriae exports through the Sec apparatus many extracellular proteins that include the key virulence factors diphtheria toxin and the adhesive pili. How these proteins attain their native conformations after translocation as unfolded precursors remains elusive. The fact that the majority of these exported proteins contain multiple cysteine residues and that several membrane-bound oxidoreductases are encoded in the corynebacterial genome suggests the existence of an oxidative protein-folding pathway in this organism. Here we show that the shaft pilin SpaA harbors a disulfide bond in vivo and alanine substitution of these cysteines abrogates SpaA polymerization and leads to the secretion of degraded SpaA peptides. We then identified a thiol-disulfide oxidoreductase (MdbA), whose structure exhibits a conserved thioredoxin-like domain with a CPHC active site. Remarkably, deletion of mdbA results in a severe temperature-sensitive cell division phenotype. This mutant also fails to assemble pilus structures and is greatly defective in toxin production. Consistent with these defects, the ΔmdbA mutant is attenuated in a guinea pig model of diphtheritic toxemia. Given its diverse cellular functions in cell division, pilus assembly and toxin production, we propose that MdbA is a component of the general oxidative folding machine in C. diphtheriae., (© 2015 John Wiley & Sons Ltd.)

Here we report the solution structure of oxidized ERp18 as determined using NMR spectroscopy. ERp18 is the smallest member of the protein disulfide isomerase (PDI) family of proteins to contain a Cys-Xxx-Xxx-Cys active site motif. It is an 18 kDa endoplasmic reticulum resident protein with unknown function although sequence similarity to individual domains of the thiol-disulfide oxidoreductase PDI suggests ERp18 may have a similar structure and function. Like the catalytic domains of PDI, ERp18 adopts a thioredoxin fold with a thioredoxin-like active site located at the N-terminus of a long kinked helix that spans the length of the protein. Comparison of backbone chemical shifts for oxidized and reduced ERp18 shows the majority of residues possess the same backbone conformation in both states, with differences limited to the active site and regions in close proximity. S(2) order parameters from NMR backbone dynamics were found to be 0.81 for oxidized and 0.91 for reduced ERp18, and these observations, in combination with amide hydrogen exchange rates, imply a more rigid and compact backbone for the reduced structure. These observations support a putative role for ERp18 within the cell as an oxidase, introducing disulfide bonds to substrate proteins, providing structural confirmation of ERp18's role as a thiol-disulfide oxidoreductase.

Methanosarcina acetivorans, a strictly anaerobic methane-producing species belonging to the domain Archaea, contains a gene cluster annotated with homologs encoding oxidative stress proteins. One of the genes (MA3736) is annotated as a gene encoding an uncharacterized carboxymuconolactone decarboxylase, an enzyme required for aerobic growth with aromatic compounds by species in the domain Bacteria. Methane-producing species are not known to utilize aromatic compounds, suggesting that MA3736 is incorrectly annotated. The product of MA3736, overproduced in Escherichia coli, had protein disulfide reductase activity dependent on a C(67)XXC(70) motif not found in carboxymuconolactone decarboxylase. We propose that MA3736 be renamed mdrA (methanosarcina disulfide reductase). Further, unlike carboxymuconolactone decarboxylase, MdrA contained an Fe-S cluster. Binding of the Fe-S cluster was dependent on essential cysteines C(67) and C(70), while cysteines C(39) and C(107) were not required. Loss of the Fe-S cluster resulted in conversion of MdrA from an inactive hexamer to a trimer with protein disulfide reductase activity. The data suggest that MdrA is the prototype of a previously unrecognized protein disulfide reductase family which contains an intermolecular Fe-S cluster that controls oligomerization as a mechanism to regulate protein disulfide reductase activity.

A potential role in disulfide bond formation in the intracellular proteins of thermophilic organisms has recently been ascribed to a new family of protein disulfide oxidoreductases (PDOs). We report on the characterization of SsPDO, isolated from the hyperthermophilic archaeon Sulfolobus solfataricus. SsPDO was cloned and expressed in Escherichia coli. We revealed that SsPDO is the substrate of a thioredoxin reductase in S. solfataricus (K(M) 0.3 microm) and not thioredoxins (TrxA1 and TrxA2). SsPDO/S. solfataricus thioredoxin reductase constitute a new thioredoxin system in aerobic thermophilic archaea. While redox (reductase, oxidative and isomerase) activities of SsPDO point to its central role in the biochemistry of cytoplasmic disulfide bonds, chaperone activities also on an endogenous substrate suggest a potential role in the stabilization of intracellular proteins. Northern and western analysis have been performed in order to analyze the response to the oxidative stress.

CcmG/DsbE is a typical thiol/disulfide oxidoreductase, exhibiting a specific reducing activity in a highly oxidizing environment, and is involved in electron transfer during the maturation of c-type cytochromes. Escherichia coli CcmG/DsbE (residues 19-185) has been crystallized using the hanging-drop vapour-diffusion technique. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 35.48, b = 48.52, c = 84.78 A. X-ray data have been collected to 1.9 A resolution.

Human erythrocyte glutathione disulfide reductase was purified using serially connected 2',5'-ADP-Sepharose 4B affinity and anion-exchange columns. About 11,000-fold purification was achieved with 90% yield. The specific activity of the final preparation was 140 units per milligram of protein. The purified enzyme gave a single band on both native and SDS-PAGE with a subunit mass of 58 kDa. Its pH optimum was 7.20. The Michaelis constants determined at pH 7.4, 37 degrees C, fell within the range of previously reported values [K(m)(NADPH) = 18 microM, at 30-200 microM NADPH; K(m)(GSSG) = 72 microM, at 40-1000 microM glutathione disulfide, both at saturating concentrations of the second substrate]. The affinity eluent NADPH and its oxidized form NADP+ were successfully removed from the enzyme on the ion-exchange column. The purification method developed is very useful when the enzyme source material is scarce (e.g., in preparations from human tissues) and may find further application in the purification of other NAD(P)H-dependent enzymes which might be inactivated by their affinity eluent(s).

A 58 kDa selenium-labeled protein purified from mouse mammary epithelial cells (MMEC) was used to examine whether selenium modulates protein synthesis or is just a marker for cellular selenium status. The protein was isolated using Sephadex G150 gel filtration and DEAE-Sephadex A50 ion-exchange chromatography. It was further analysed using 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and was found as a single spot with a pI of 4.6. The immunoreactivity with anti-58 kDa antiserum and the 75Se signal co-localized on a single 58 kDa protein band on both 1D- and 2D-PAGE. Partial amino acid analysis of the peptide showed homology with the thiol protein disulfide oxidoreductase (TPDO). Varying the selenium concentration in culture medium did not affect the protein content or the immunoreactivity of the 58 kDa protein. Additionally, selenium did not seem to regulate the activity of TPDO in TM6 cells. The glutathione peroxidase activity of TM6 cells, taken as the internal positive control, was enhanced with the increase in selenium concentration in the medium. The results suggest that selenium is attached to the 58 kDa protein, but does not regulate either its protein synthesis or its functional activity. We conclude that selenium labeling of the 58 kDa protein reflects the cellular selenium status but probably is not involved in its chemopreventive ability.

Proteins of the internal nuclear matrix from chicken liver were fractionated, by chromatographic procedures, in non denaturing conditions. At least two fractions were present with phosphatidylinositol-specific phospholipase C and three with thiol:protein-disulfide oxidoreductase activity. A 57kDa protein was isolated which copurified with both these activities. Partial amino acid sequences showed a high degree of homology with a cytosolic protein previously identified as a phospholipase C and with a microsomal protein identified as a thiol:protein-disulfide oxidoreductase. Our finding leaves the question still unanswered of the real function of this protein, which for the first time has been isolated from the nuclear matrix.

Thiol:protein-disulfide oxidoreductase catalyzes the GSH reduction of protein disulfides to sulfhydryls. Chromatography of solubilized hepatic microsomes on Mono Q yielded two peaks, Q-2 and Q-5, which contained all the thiol:protein-disulfide oxidoreductase activity. These were further purified by chromatofocusing giving specific activities of 14.4 and 45.9 nmol/mg of protein/min, respectively with purifications of 45.0- and 143.6-fold. Amino acids 1-18 of Q-5 were the same as previously reported for Thiol:protein-disulfide oxidoreductase (Edman, J. C., Ellis, L., Blacher, R. W., Roth, R. A., and Rutter, W. J. (1985) Nature 317, 267-270), except amino acid 1 was leucine instead of aspartate and amino acid 6 was asparagine instead of glutamate. The N-terminal amino acid sequence of Q-2 differed markedly from Q-5 but Q-2 showed 100% identity at amino acids 25-54, 258-269, 285-310, 347-350, 412-419, and 434-463 for the reported sequence of rat, hepatic, cytosolic phosphatidylinositol-specific phospholipase C form 1a (PLC) (Bennett, C. F., Balcarek, J. M., Varrichio, A., and Crooke, S. T. (1988) Nature 334, 268-270). PLC activity was found in the elution from the Mono Q column, but none was found in purified Q-2 or Q-5. Antibodies to Q-5 reacted with Q-2, but anti-Q-2 did not react with Q-5. Anti-Q-2 antibody showed immunoreactivity with 55- and 60-kDa microsomal proteins, whereas Q-5 antibody reacted with a number of microsomal proteins. Although Q-2 was immunoreactive with a polyclonal antibody to guinea pig, uterine cytosolic PLC, partially purified PLCs from rat liver cytosol did not react to this antibody. Our data would suggest that the published sequence for PLC form 1a may actually be the sequence for Q-2.

1. Protein disulphide-isomerase (EC 5.3.4.1) and glutathione-insulin transhydrogenase (EC 1.8.4.2) activities in bovine liver were studied in parallel during purification of 'thiol-protein disulphide oxidoreductase' by the procedure of Carmichael, Morin & Dixon [(1977) J Biol. Chem. 252, 7163-7167]. The two activities showed no quantitative co-purification and were partially resolved by (NH4)SO4 precipitation, indicating that distinct enzymes are present. 2. Protein disulphide-isomerase was purified by a relatively rapid method involving a combination of the early stages of the Carmichael procedure and covalent chromatography, with a new stepwise elution procedure. Ion-exchange chromatography yields a homogeneous preparation of mol.wt. 57 000. 3. The relationship between protein disulphide-isomerase, glutathione-insulin transhydrogenase and 'thiol-protein disulphide oxidoreductase' is discussed.

Thiole: Protein disulfide Oxidoreductase (E. C. 1.8.4.2) is capable of catalyzing thiole-disulfid exchange reactions and might have an important function in both protein biosynthesis and degradation. By using histochemical methods we were able to demonstrate the localization of this enzyme in the Langerhans'islets and in the acinus cells of rat pancreas. The reaction of the acinus cells, however, was much weaker than that of the islets. Electron microscopic experiments revealed the enzyme to be located in the outer membrane of the nucleus, in the membranes of the endoplasmic reticulum and B-cell granules, and in the plasmalemma also. All these structures are known to be involved in the insulin biosynthesis and secretion. There is a good correlation between morphological and biochemical findings. In acinus cells there are a few reaction in the outer nucleus membrane, the membranes of the endoplasmatic reticulum, and the plasmalemm.

A rapid purification procedure of thiol:protein-disulfide oxidoreductase (EC 1.8.4.2) from bovine liver has been developed. The procedure is based on that of D. F. Carmichael, J. E. Morin, and J. E. Dixon (1977, J. Biol. Chem. 252, 7163-7167), and contains the following steps: homogenization in Triton X-100, selective heat denaturation, chromatography on CM-Sephadex C-50, and chromatography on DEAE-Sephadex A-50. The final preparation has a high specific activity and a high level of purity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Rat liver thiol:protein-disulfide oxidoreductase/glutathione-insulin transhydrogenase (glutathione:protein disulfide oxidoreductase, EC 1.8.4.2) was purified and found to give two bands on sodium dodecyl sulfate polyacrylamide gel electrophoresis. A monoclonal antibody was produced against this enzyme preparation and found to remove all the insulin degrading activity of purified preparations of the enzyme. This monoclonal antibody was also found to react with the two different forms of the enzyme observed on gel electrophoresis. These results suggest that glutathione-insulin transhydrogenase can exist in more than one state.

A thiol:protein disulfide oxidoreductase which degrades insulin into its A and B chains has been purified to homogeneity from bovine liver. The initial extraction procedure used 1% Triton X-100, which increased the soluble insulin degrading activity 100 to 800% as a result of solubilizing a membrane-associated enzyme. The criteria for homogeneity of the isolated protein include sodium dodecyl sulfate and disc gel electrophoresis. Characterization of the homogeneous protein by sedimentation equilibrium centrifugation indicates that a single protein is present which has a mass of 60,000 daltons. A single amino-terminal end group and molecular weight estimation by sodium dodecyl sulfate gel electrophoresis indicated that the thiol:protein disulfide oxidoreductase consists of a single polypeptide chain. The pure enzyme was also examined by gel filtration chromatography and has an apparent molecular weight of 92,000. The amino acid composition and carbohydrate content were also determined. The carbohydrate analysis demonstrated the presence of D-mannose, D-galactose, 2-acetamido-2-deoxy-D-glucose, and N-acetylneuraminic acid which comprise a total of 12% by weight of the isolated protein.

Elution of complex protein mixtures on a matrix containing reactive disulphide bonds (Thiopropyl-Sepharose 6B, Pharmacia) results in immobilisation of thiol-containing molecules. Specific protein fractions can be displaced from the gel using different low-molecular-weight reducing agents. Thus a single sequential elution can separate and resolve thiol-containing proteins in a rapid and convenient step. The method is illustrated with reference to beef liver thiol : disulphide oxidoreductases.

Insulin degrading enzymes of rat liver cytosol, the so-called insulin and glucagon degrading proteinase (IGP, EC 3.4.23.5), and two forms of the insulin degrading thiol-protein-disulfide oxidoreductase/isomerase (glutathione-insulin transhydrogenase, TPO, EC 1.8.4.2/5.3.4.1) were separated from each other and partially purified on DEAE-Sephadex. The highly purified proteinase was obtained by polyacrylamide gel electrophoresis of the DEAE-Sephadex-purified enzyme fraction and was used to produce monospecific antibodies to the IGP in rabbits. Strong evidence is given that the insulin and glucagon degrading proteinase is an autonomous enzyme existing in addition to the TPO forms in the cytosol of the liver. Combined action of the proteinase and the TPO system on radioiodinated insulin under various conditions in vitro revealed an independent and non-sequential degradation of insulin by these two enzyme systems.

1. Protein disulphide-isomerase (EC 5.3.4.1) and glutathione-insulin transhydrogenase (EC 1.8.4.2) were resolved by covalent chromatography. Both activities, in a partially purified preparation from bovine liver, bind covalently as mixed disulphides to activated thiopropyl-Sepharose 6B, in a new stepwise elution procedure protein disulphide-isomerase is displaced in mildly reducing conditions whereas glutathione-insulin transhydrogenase is only displaced by more extreme reducing conditions. 2. This together with evidence for partial resolution of the two activities by ion-exchange chromatography, conclusively establishes that the two activities are not alternative activities of a single bovine liver enzyme. 3. Protein disulphide-isomerase, partially purified by a published procedure, has now been further purified by covalent chromatography and ion-exchange chromatography. The final material is 560-fold purified relative to a bovine liver homogenate; it has barely detectable glutathione-insulin transhydrogenase activity. 4. The purified protein disulphide-isomerase shows a single major band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis corresponding to a mol.wt. of 57000. 5. The purified protein disulphide-isomerase has Km values for 'scrambled' ribonuclease and dithiothreitol of 23 microgram/ml and 5.4 microM respectively and has a sharp pH optimum at 7.5. The enzyme has a broad thiol-specificity, and several monothiols, at 1mM, can replace dithiothreitol. 6. The purified protein disulphide-isomerase is completely inactivated after incubation with a 2-3 fold molar excess of iodoacetate. The enzyme is also significantly inhibited by low concentrations of Cd2+ ions. These findings strongly suggest the existence of a vicinal dithiol group essential for enzyme activity. 7. When a range of thiols were used as co-substrates for protein disulphide-isomerase activity, the activities were found to co-purify quantitatively, implying the presence of a single protein disulphide-isomerase of broad thiol-specificity. Glutathione-disulphide transhydrogenase activities, assayed with a range of disulphide compounds, did not co-purify quantitatively.

In the presence of thiols such as glutathione or dithiothreitol, choleragen catalyzes the NAD-dependent ADP-ribosylation of arginine and proteins; thiols reduce the disulfide linking the A1 and A2 peptides of the A protomer of the toxin, releasing the active A1 peptide. Homogeneous thiol:protein disulfide oxidoreductase from bovine liver, in the presence of limiting concentrations of glutathione or dithiothreitol, increased the rate of activation of choleragen and its A protomer. The ability of oxidoreductase preparations to activate choleragen co-chromatographed with oxidoreductase protein on gel permeation columns and was proportional to the concentration of oxidoreductase in the assay. In the presence of oxidoreductase, the concentrations of glutathione and dithiothreitol necessary for activation were reduced. Thiol:protein disulfide oxidoreductase could play a role in the reduction of choleragen and release of the catalytically active A1 peptide.

Thiol:protein disulfide oxidoreductase activity was assayed in extracts of cultured normal human skin fibroblasts. Enzyme activity in confluent fibroblasts was dependent on growth conditions. In serum-deprived fibroblasts grown in minimal medium enzyme activity was approximately 40% of that observed in fibroblasts maintained in medium supplemented with 10% fetal calf serum. In fibroblasts cultured in medium supplemented only with insulin, activity was 35% greater than that in fibroblasts cultured in unsupplemented defined medium. Antibodies raised against purified bovine liver thiol:protein disulfide oxidoreductase immunoprecipitated all of the activity present in fibroblast extracts. The thiol:protein disulfide oxidoreductase from human fibroblasts thus appears to share antigenic determinants with the bovine liver enzyme. The human fibroblast may serve as an in vitro model to study the regulation of the oxidoreductase.

1. Protein disulphide-isomerase and glutathione-insulin transhydrogenase activities were assayed in parallel through a conventional purification of protein disulphide-isomerase from ox liver. 2. Throughout a series of purification steps (differential centrifugation, acetone extraction, (NH4)2SO4 precipitation and ion-exchange chromatography), the two activities appeared in the same fractions but were purified to different extents. 3. The final sample was 143-fold purified in protein disulphide-isomerase but only 10-fold purified in glutathione-insulin transhydrogenase; nevertheless the two activities in this preparation were not resolved by high-resolution isoelectric focusing and both showed pI4.65. 4. In a partially purified preparation containing both activities, glutathione-insulin transhydrogenase was far more sensitive to heat denaturation than was protein disulphide-isomerase; conversely protein disulphide-isomerase was more sensitive to inactivation by deoxycholate. 5. The data are inconsistent with a single enzyme being responsible for all the protein disulphide-isomerase and glutathione-insulin transhydrogenase activity of ox liver. It is suggested that several similiar thiol-protein disulphide oxidoreductases of overlapping specificities may better account for the data.