Vibrio cholerae periplasmic superoxide dismutase: isolation of the gene and overexpression of the protein

Superoxide dismutases are ubiquitous enzymes which play an important role in protecting cells against oxidative damage and which have also been shown to contribute to the pathogenicity of many bacterial species. Here we demonstrate that Vibrio cholerae, the causative agent of cholerae, expresses an active periplasmic Cu,Zn superoxide dismutase. Moreover, we have set up an expression system yielding large amounts of V. cholerae recombinant Cu,Zn superoxide dismutase in the periplasm of Escherichia coli and a procedure to obtain the enzyme in a highly purified form. Unlike the bovine enzyme, V. cholerae Cu,Zn superoxide dismutase has been proved to be highly resistant to inactivation by hydrogen peroxide. This property, which appears to be common to other bacterial enzymes of this class, might improve the ability of Cu,Zn superoxide dismutase to protect bacteria against the reactive oxygen species produced by phagocytes.     

A Primary Role for Disulfide Formation in the Productive Folding of Prokaryotic Cu,Zn-superoxide Dismutase

Enzymatic activation of Cu,Zn-superoxide dismutase (SOD1) requires not only binding of a catalytic copper ion but also formation of an intramolecular disulfide bond. Indeed, the disulfide bond is completely conserved among all species possessing SOD1; however, it remains obscure how disulfide formation controls the enzymatic activity of SOD1. Here, we show that disulfide formation is a primary event in the folding process of prokaryotic SOD1 (SodC) localized to the periplasmic space. Escherichia coli SodC was found to attain β-sheet structure upon formation of the disulfide bond, whereas disulfide-reduced SodC assumed little secondary structure even in the presence of copper and zinc ions. Moreover, reduction of the disulfide bond made SodC highly susceptible to proteolytic degradation. We thus propose that the thiol-disulfide status in SodC controls the intracellular stability of this antioxidant enzyme and that the oxidizing environment of the periplasm is required for the enzymatic activation of SodC.     

Friction-Limited Folding of Disulfide-Reduced Monomeric SOD1

The folding reaction of a stable monomeric variant of Cu/Zn superoxide dismutase (mSOD1), an enzyme responsible for the conversion of superoxide free radicals into hydrogen peroxide and oxygen, is known to be among the slowest folding processes that adhere to two-state behavior. The long lifetime, ∼10 s, of the unfolded state presents ample opportunities for the polypeptide chain to transiently sample nonnative structures before the formation of the productive folding transition state. We recently observed the formation of a nonnative structure in a peptide model of the C-terminus of SOD1, a sequence that might serve as a potential source of internal chain friction-limited folding. To test for friction-limited folding, we performed a comprehensive thermodynamic and kinetic analysis of the folding mechanism of mSOD1 in the presence of the viscogens glycerol and glucose. Using a, to our knowledge, novel analysis of the folding reactions, we found the disulfide-reduced form of the protein that exposes the C-terminal sequence, but not its disulfide-oxidized counterpart that protects it, experiences internal chain friction during folding. The sensitivity of the internal friction to the disulfide bond status suggests that one or both of the cross-linked regions play a critical role in driving the friction-limited folding. We speculate that the molecular mechanisms giving rise to the internal friction of disulfide-reduced mSOD1 might play a role in the amyotrophic lateral sclerosis-linked aggregation of SOD1.     

Folding of Cu, Zn superoxide dismutase and familial amyotrophic lateral sclerosis

Cu, Zn superoxide dismutase (SOD1) has been implicated in the familial form of the neurodegenerative disease amyotrophic lateral sclerosis (ALS). It has been suggested that mutant mediated SOD1 misfolding/aggregation is an integral part of the pathology of ALS. We study the folding thermodynamics and kinetics of SOD1 using a hybrid molecular dynamics approach. We reproduce the experimentally observed SOD1 folding thermodynamics and find that the residues which contribute the most to SOD1 thermal stability are also crucial for apparent two-state folding kinetics. Surprisingly, we find that these residues are located on the surface of the protein and not in the hydrophobic core. Mutations in some of the identified residues are found in patients with the disease. We argue that the identified residues may play an important role in aggregation. To further characterize the folding of SOD1, we study the role of cysteine residues in folding and find that non-native disulfide bond formation may significantly alter SOD1 folding dynamics and aggregation propensity.     

Cloning,production and characterisation of a recombinant Cu/Zn superoxide dismutase from Taenia solium

A full-length complementary DNA clone encoding a cytosolic Cu/Zn superoxide dismutase with a M(r) of 15,588 Da was isolated from a Taenia solium larvae complementary DNA library. Comparison analysis of its deduced amino acid sequence revealed a 71% identity with Schistosoma mansoni, 57.2-59.8% with mammalian and less than 54% with other helminth cytosolic Cu/Zn superoxide dismutase. The characteristic motifs and the amino acid residues involved in coordinating copper and zinc enzymatic function are conserved. The T. solium Cu/Zn superoxide dismutase was expressed in the pRSET vector. Enzymatic and filtration chromatographic analysis showed a recombinant enzyme with an activity of 2,941 U/mg protein and a native M(r) of 37 kDa. Inhibition assays using KCN, H(2)O(2), NaN(3) and SDS indicated that Cu/Zn is the metallic cofactor in the enzyme. Thiabendazole (500 microM) and albendazole (300 microM) completely inhibited the activity of T. solium Cu/Zn superoxide dismutase. Thiabendazole had no effect on bovine Cu/Zn superoxide dismutase; in contrast, albendazole had a moderate effect on it at same concentrations. Antibodies against T. solium Cu/Zn superoxide dismutase did not affect the enzymatic function; nevertheless, it cross reacts with several Taenia species, but not with trematodes, nematodes, pig, human and bovine Cu/Zn superoxide dismutase enzymes. Western blot analysis indicated the enzyme was expressed in all stages. These results indicate that T. solium possesses a Cu/Zn superoxide dismutase enzyme that can protect him from oxidant-damage caused by the superoxide anion.     

The nonconsecutive disulfide bond of Escherichia coli phytase (AppA) renders it dependent on the protein-disulfide isomerase, DsbC

The formation of protein disulfide bonds in the Escherichia coli periplasm by the enzyme DsbA is an inaccurate process. Many eukaryotic proteins with nonconsecutive disulfide bonds expressed in E. coli require an additional protein for proper folding, the disulfide bond isomerase DsbC. Here we report studies on a native E. coli periplasmic acid phosphatase, phytase (AppA), which contains three consecutive and one nonconsecutive disulfide bonds. We show that AppA requires DsbC for its folding. However, the activity of an AppA mutant lacking its nonconsecutive disulfide bond is DsbC-independent. An AppA homolog, Agp, a periplasmic acid phosphatase with similar structure, lacks the nonconsecutive disulfide bond but has the three consecutive disulfide bonds found in AppA. The consecutively disulfide-bonded Agp is not dependent on DsbC but is rendered dependent by engineering into it the conserved nonconsecutive disulfide bond of AppA. Taken together, these results provide support for the proposal that proteins with nonconsecutive disulfide bonds require DsbC for full activity and that disulfide bonds are formed predominantly during translocation across the cytoplasmic membrane.     

Periplasmic competition for zinc uptake between the metallochaperone ZnuA and Cu,Zn superoxide dismutase

We have investigated the availability of zinc in the periplasmic space of Escherichia coli using a mutant Cu,Zn superoxide dismutase whose dimerization is triggered by zinc binding. This mutant enzyme accumulates in the monomeric form when wild type cells are grown in minimal medium, but assembles in the dimeric form when it is produced in the same medium by a mutant strain lacking the periplasmic zinc metallochaperone ZnuA. These results indicate that periplasmic zinc-containing proteins compete for metal binding when bacteria grow in environments where this element is present in traces. The effective ZnuA ability to sequester the available zinc ions from the periplasm suggests that zinc-containing cytoplasmic proteins are more important for bacterial viability than the periplasmic ones.     

Ozone-induced inactivation of antioxidant enzymes

Ozone is an air pollutant that damages a variety of biomolecules. We investigated ozone-induced inactivation of three major antioxidant enzymes. Cu/Zn superoxide dismutase was inactivated by ozone in a concentration-dependent manner. The concentration of ozone for 50% inactivation was approximately 45 microM when 10 microM Cu/Zn superoxide dismutase was incubated for 30 min in the presence of ozone. SDS-polyacrylamide gel electrophoresis (PAGE) showed that the enzyme was randomly fragmented. Both ascorbate and glutathione were very effective in protecting Cu/Zn superoxide dismutase from ozone-induced inactivation. The other two enzymes, catalase and glutathione peroxidase, were much more resistant to ozone than Cu/Zn superoxide dismutase. The ozone concentrations for 50% inactivation of 10 microM catalase and glutathione peroxidase were 500 and 240 microM, respectively. SDS-PAGE demonstrated that ozone caused formation of high molecular weight aggregates in catalase and dimerization in glutathione peroxidase. Glutathione protected catalase and glutathione peroxidase from ozone but the effective concentrations were much higher than that for Cu/Zn superoxide dismutase. Ascorbate was almost ineffective. The result suggests that, among the three antioxidant enzymes, Cu/Zn superoxide dismutase is a major target for ozone-induced inactivation and both glutathione and ascorbate are very effective in protecting the enzyme from ozone.     

Mechanism and Atomic Structure of Superoxide Dismutase

The active site Cu ion in Cu,Zn superoxide dismutase is alternately oxidized and reduced during the enzymatic dismutation of superoxide to hydrogen peroxide and molecular oxygen. For oxidized Cu,Zn superoxide dismutase, an atomic structure has been determined for the human enzyme at 2.5 A resolution. The resolution of the bovine enzyme structure has been extended to 1.8 A. Atomic resolution data has been, collected for reduced and inhibitor-bound Cu,Zn superoxide dismutases. and the interpretation of the' electron density difference maps is in progress. The geometry and molecular surfaces of the active sites in these structures, together with biochemical data, suggest a specific model for the enzyme mechanism. Similarities in the active site geometry of the Mn and Fe superoxide dismutases with the Cu.Zn enzyme suggest that dismutation in these enzymes may follow a similar mechanism.     

Evolutionary Aspects of Superoxide Dismutase: The Copper/Zinc Enzyme

Copper/zinc superoxide dismutase is typically an enzyme of eukaryotes. The presence of the enzyme in the ponyfish symbiont Photobocterium leiognothi and some free living bacteria does not have an immediate explanation. Amino acid sequence alignment of 19 Cu/Zn superoxide disrnutases shows 21 invariant residues in key positions related to maintenance of the β-barrel fold, the active site structure including the electrostatic channel loop, and dimer contacts. Nineteen other residues are invariant in 18 of the 19 sequences. Thirteen of these nearly invariant residues show substitutions in Photobocterium Cu/Zn superoxide dismutase. Copper/zinc superoxide disrnutase from the trematode Schisiosoma mansoni shows an N-terminal sub-domain with a hydrophobic leader peptide. as in human extracellular superoxide dismutase which is a Cu/Zn enzyme. The latter also has a C-terminal sub-domain with preponderance of hydrophilic and positively charged residues. The amino acid sequence of this superoxide dismutase between the N-terminal and C-terminal regions shares many features of cytosolic Cu/Zn superoxide dismutase. including 20 of the 21 invariant residues found in 19 Cu/Zn enzymes, suggesting a similar type of β-barrel fold and active site structure for the extracellular enzyme.     

Biosynthesis and Cellular Distribution of the Two Superoxide Dismutases of Dactylium dendroides

The synthesis and subcellular localization of the two superoxide dismutases of Dactylium dendroides were studied in relation to changes in copper and manganese availability. Cultures grew normally at all medium copper concentrations used (10 nM to 1 mM). In the presence of high (10 μM) copper, manganese was poorly absorbed in comparison to the other metals in the medium. However, cells grown at 10 nM copper exhibited a 3.5-fold increase in manganese content, while the concentration of the other metals remained constant. Cultures grown at 10 nM copper or more had 80% Cu/Zn enzyme and 20% mangani enzyme; the former was entirely in the cytosol, and the latter was mitochondrial. Removal of copper from the medium resulted in decreased Cu/Zn superoxide dismutase synthesis with a concomitant increase in the mangani enzyme such that total cellular superoxide dismutase activity remained constant. The mangani enzyme in excess of the 20% was present in the non-mitochondrial fraction. The mitochondria, therefore, show no variability with respect to superoxide dismutase content, whereas the soluble fraction varies from 100 to 13% Cu/Zn superoxide dismutase. Copper-starved cells that were synthesizing predominantly mangani superoxide dismutase could be switched over to mostly Cu/Zn superoxide dismutase synthesis by supplementing the medium with copper during growth. Immunoprecipitation experiments suggest that the decrease in Cu/Zn activity at low copper concentration is a result of decreased synthesis of that protein rather than the production of an inactive apoprotein.     

Disulfide-Reduced ALS Variants of Cu, Zn Superoxide Dismutase Exhibit Increased Populations of Unfolded Species

Cu,Zn superoxide dismutase (SOD1) is a dimeric metal-binding enzyme responsible for the dismutation of toxic superoxide to hydrogen peroxide and oxygen in cells. Mutations at dozens of sites in SOD1 induce amyotrophic lateral sclerosis (ALS), a fatal gain-of-function neurodegenerative disease whose molecular basis is unknown. To obtain insights into effects of the mutations on the folded and unfolded populations of immature monomeric forms whose aggregation or self-association may be responsible for ALS, the thermodynamic and kinetic folding properties of a set of disulfide-reduced and disulfide-oxidized Zn-free and Zn-bound stable monomeric SOD1 variants were compared to properties of the wild-type (WT) protein. The most striking effect of the mutations on the monomer stability was observed for the disulfide-reduced metal-free variants. Whereas the WT and S134N monomers are > 95% folded at neutral pH and 37 °C, A4V, L38V, G93A, and L106V ranged from 50% to ∼ 90% unfolded. The reduction of the disulfide bond was also found to reduce the apparent Zn affinity of the WT monomer by 750-fold, into the nanomolar range, where it may be unable to compete for free Zn in the cell. With the exception of the S134N metal-binding variant, the Zn affinity of disulfide-oxidized SOD1 monomers showed little sensitivity to amino acid replacements. These results suggest a model for SOD1 aggregation where the constant synthesis of ALS variants of SOD1 on ribosomes provides a pool of species in which the increased population of the unfolded state may favor aggregation over productive folding to the native dimeric state.     

Functional and crystallographic characterization of Salmonella typhimurium Cu,Zn superoxide dismutase coded by the sodCI virulence gene

The functional and three-dimensional structural features of Cu,Zn superoxide dismutase coded by the Salmonella typhimurium sodCI gene, have been characterized. Measurements of the catalytic rate indicate that this enzyme is the most efficient superoxide dismutase analyzed so far, a feature that may be related to the exclusive association of the sodCI gene with the most pathogenic Salmonella serotypes. The enzyme active-site copper ion is highly accessible to external probes, as indicated by quenching of the water proton relaxation rate upon addition of iodide. The shape of the electron paramagnetic resonance spectrum is dependent on the frozen or liquid state of the enzyme solution, suggesting relative flexibility of the copper ion environment. The crystal structure (R-factor 22.6%, at 2.3 A resolution) indicates that the dimeric enzyme adopts the quaternary assembly typical of prokaryotic Cu,Zn superoxide dismutases. However, when compared to the structures of the homologous enzymes from Photobacterium leiognathi and Actinobacillus pleuropneumoniae, the subunit interface of Salmonella Cu,Zn superoxide dismutase shows substitution of 11 out of 19 interface residues. As a consequence, the network of structural water molecules that fill the dimer interface cavity is structured differently from the other dimeric bacterial enzymes. The crystallographic and functional characterization of this Salmonella Cu,Zn superoxide dismutase indicates that structural variability and catalytic efficiency are higher in prokaryotic than in the eukaryotic homologous enzymes.     

The effect of bacterial endotoxin on synthesis of (Cu,Zn)superoxide dismutase in lungs of oxygen-exposed rats

Administration of bacterial endotoxin to rats exposed to greater than 95% O2 results in increased lung superoxide dismutase activity, decreased O2-induced lung damage, and a 3- to 4-fold improvement in survival rate (Frank, L., Yam, J., and Roberts, R. J. (1978) J. Clin. Invest, 61, 269-275). Antibodies to rat liver (Cu,Zn) superoxide dismutase were prepared and utilized to investigate the mechanism by which endotoxin treatment leads to increased lung superoxide dismutase activity. Assay of enzyme activity and of immunodetectable enzyme showed that the increased activity is due to an increase in the number of enzyme molecules rather than activation of existing enzyme. Compared to air controls, lung slices from rats exposed to greater than 95% O2 and treated with endotoxin have elevated rats of synthesis of (Cu,Zn)superoxide dismutase (51%) and of total protein (100%). Lung slices from untreated rats exposed to greater than 95% O2 have no such elevations. Endotoxin treatment thus appears to stimulate lung protein synthesis, leading to greater (Cu,Zn)superoxide dismutase activity due to an increased number of enzyme molecules.     

Inactive extracellular superoxide dismutase disrupts secretion and function of active extracellular superoxide dismutase

Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme that protects cells and tissues from extracellular damage by eliminating superoxide anion radicals produced during metabolism. Two different forms of EC-SOD exist, and their different enzyme activities are a result of different disulfide bond patterns. Although only two folding variants have been discovered so far, five folding variants are theoretically possible. Therefore, we constructed five different mutant EC-SOD expression vectors by substituting cysteine residues with serine residues and evaluated their expression levels and enzyme activities. The mutant EC-SODs were expressed at lower levels than that of wild-type EC-SOD, and all of the mutants exhibited inhibited extracellular secretion, except for C195S ECSOD. Finally, we demonstrated that co-expression of wild-type EC-SOD and any one of the mutant EC-SODs resulted in reduced secretion of wild-type EC-SOD. We speculate that mutant EC-SOD causes malfunctions in systems such as antioxidant systems and sensitizes tissues to ROS-mediated diseases.     

Differential temperature sensitivity of pea superoxide dismutases

The activity of pea (Pisum sativum L.) Cu/Zn and Mn superoxide dismutase isoforms was evaluated across a range of temperatures from 10 to 45°C. Maximal activity of the Cu/Zn and Mn superoxide dismutase isoforms was observed at 10°C. Both cytoplasmic and chloroplast Cu/Zn superoxide dismutases exhibit a reduction in staining intensity with increasing temperatures. Mn superoxide dismutase, however, maintained a relatively constant staining intensity across the range of temperatures evaluated. An unrelated enzyme used as a control, malate dehydrogenase, exhibited the expected increase in staining activity with increasing temperatures. These results describe a unique response of a protection enzyme to temperature.     

Periplasmic Cu,Zn superoxide dismutase and cytoplasmic Dps concur in protecting Salmonella enterica serovar Typhimurium from extracellular reactive oxygen species

Several bacteria possess periplasmic Cu,Zn superoxide dismutases which can confer protection from extracellular reactive oxygen species. Thus, deletion of the sodC1 gene reduces Salmonella enterica serovar Typhimurium ability to colonize the spleens of wild type mice, but enhances virulence in p47phox mutant mice. To look into the role of periplamic Cu,Zn superoxide dismutase and into possible additive effects of the ferritin-like Dps protein involved in hydrogen peroxide detoxification, we have analyzed bacterial survival in response to extracellular sources of superoxide and/or hydrogen peroxide. Exposure to extracellular superoxide of Salmonella Typhimurium mutant strains lacking the sodC1 and sodC2 genes and/or the dps gene does not cause direct killing of bacteria, indicating that extracellular superoxide is poorly bactericidal. In contrast, all mutant strains display a sharp hydrogen peroxide-dependent loss of viability, the dps,sodC1,sodC2 mutant being less resistant than the dps or the sodC1,sodC2 mutants. These findings suggest that the role of Cu,Zn superoxide dismutase in bacteria is to remove rapidly superoxide from the periplasm to prevent its reaction with other reactive molecules. Moreover, the nearly additive effect of the sodC and dps mutations suggests that localization of antioxidant enzymes in different cellular compartments is required for bacterial resistance to extracytoplasmic oxidative attack.     

铜锌超氧化物歧化酶(SOD)研究进展——从基因到功能

超氧化物歧化酶(SOD,EC 1.15.1.1),己经在多种组织中发现,它能将O2.-催化生成H2O2及O2.迄今为止,已经从哺乳动物体内分离出三种SOD:CuZnSOD(SOD1)、MnSOD(SOD2)TLEC-SOD(胞外超氧化物歧化酶,SOD3),各自具有不同的生化及分子特性.CuZnSOD(SOD1),是一类含有Cu及Zn原子的二聚体,存在于特定细胞的基质内,约占SOD总量的90%.在胞质及周质中,SOD以二聚体形式存在,而在线粒体及质外,则以四聚体形式存在.在保护脑、肺及其它组织的氧化应激中,CuZnSOD被认为起着保护作用.运动神经元肌萎缩侧索硬化症(ALS),据称也与同源二聚体CuZnSOD的错误折叠有关,己经报导,有多个CuZnSOD基因位点突变与ALS有关.本文将从基因的结构、表达、调节及蛋白的结构与功能等方面,对CuZnSOD进行简要论述.     

Superoxide dismutase during glucose repression of Hansenula polymorpha CBS 4732

Hansenula polymorpha CBS 4732 was studied during cultivation on methanol and different glucose concentrations. Activities of Cu/Zn and Mn superoxide dismutase, catalase and methanol oxidase were investigated. During cultivation on methanol, increased superoxide dismutase and catalase activities and an induced methanol oxidase were achieved. Transfer of a methanol grown culture to medium with a high glucose concentration caused growth inhibition, low consumption of carbon, nitrogen and phosphate substrates, methanol oxidase inactivation as well as decrease of catalase activity (21.8 +/- 0.61 deltaE240 x min(-1) x mg protein(-1)). At the same time, a high value for superoxide dismutase enzyme was found (42.9 +/- 0.98 U x mg protein(-1), 25% of which was represented by Mn superoxide dismutase and 75% - by the Cu/Zn type). During derepression methanol oxidase was negligible (0.005 +/- 0.0001 U x mg protein(-1)), catalase tended to be the same as in the repressed culture, while superoxide dismutase activity increased considerably (63.67 +/- 1.72 U x mg protein(-1), 69% belonging to the Cu/Zn containing enzyme). Apparently, the cycle of growth inhibition and reactivation of Hansenula polymorpha CBS 4732 cells is strongly connected with the activity of the enzyme superoxide dismutase.     

Purification of a fully metal-depleted Cu, Zn superoxide dismutase from copper-deficient rat liver

A copper-deprived form of the enzyme Cu, Zn superoxide dismutase was identifiedin the liver of rats made copper-deficient by dietary restriction. In homogenates ofsuch livers Cu, Zn superoxide dismutase presents a dis-homogeneous electrophoreticprofile with respect to the native enzyme. When rat liver extracts were treated withexogenous copper an electrophoretic pattern resembling the native one was observed.Enzyme purified by chromatography on DE-52 resin shows two major components, onecorresponding to genuine, native enzyme and another one, eluting at higher ionicstrength. The latter protein (Fraction II) consists of several isoforms which showthe same characteristics of the native superoxide dismutase as far as immunoreactivityand molecular weight are concerned, but with decreased contents of copper and zinc. Itscatalytic constant, referring to copper content, was 15 times lower than that obtainedfor the native enzyme. Moreover, the catalytic power of purified Fraction II was notregained upon incubation with copper. The occurrence of a superoxide dismutase voidof metals confirms the hypothesis that this protein plays a dual physiological role:in metal metabolism and in superoxide anion dismutation.