Yeast Superoxide Dismutase Research Articles

Effect of Yeast Superoxide Dismutase Treatment on Some Mediators of Infl ammation during Adjuvant-Induced Arthritis in Mice

* Author for correspondence and reprint requests Z. Naturforsch. 65c, 141-147 (2010); received June 17/July 21, 2009 The superoxide radical (O2-), hydrogen peroxide (H2O2) and nitric oxide (NO) are pleiotropic inflammatory mediators which play an important role in inflammatory joint diseases. They are overproduced during rheumatoid arthritis and its experimental model - adjuvant-induced arthritis in rodents--and may be detected both systemically and intra-articularly. Their secretion is up-regulated by proinflammatory cytokines such as IFN-gamma, IL-12, IL-6 and TNF-alpha, and they are responsible for the destruction of joint tissue. In this work, the effect of superoxide dismutase (SOD) from a thermotolerant yeast strain, Kluyveromyces marxianus, on the production of proinflammatory cytokines, reactive oxygen and nitrogen species was studied. Mice received three intraperitoneal injections of yeast SOD at a dose of 10 mg/ kg body weight (30,000 U/kg) on consecutive days starting on the day after arthritic induction. On days 3, 8 and 14 post induction peritoneal macrophages were isolated and both spontaneous and stimulated production of reactive oxygen and nitrogen metabolites were measured. Early in arthritic development yeast SOD treatment did not influence the O2- production, but on day 14 both spontaneous and PMA-induced secretion were dramatically reduced. Spontaneous H2O2 release was inhibited on day 14, while PMA-stimulated production was decreased from the beginning of the arthritic development. Yeast SOD treatment effectively suppressed the spontaneous and recombinant mouse IFN-gamma + LPS induced release of NO as well. Serum levels of proinflammatory cytokines, IL-12, IFN-gamma, IL-6 and TNF-alpha, were also significantly reduced. The obtained results show some of the mechanisms of action of SOD in reducing the severity of arthritic inflammation. Besides direct inhibition of joint tissue destruction exogenous SOD substantially limits the existing positive feedback between secretion of reactive oxygen species and inflammatory cytokine production.

Yeast superoxide dismutase mutants reveal a pro-oxidant action of weak organic acid food preservatives

Saccharomyces cerevisiae could provide a simple experimental system for testing the antioxidant or pro-oxidant actions of chemicals, because it has the capacity for aerobic and anaerobic growth and can readily lose its mitochondrial electron transport chain (the major endogenous source of reactive oxygen species [ROS]). This study showed that yeast superoxide dismutase mutants, in a simple petri dish test, readily distinguish a compound that enhances the detrimental effects of endogenous ROS production by the mitochondrial respiratory chain from another chemical that generates oxidative stress by redox cycling. Using this system, weak organic acid food preservatives are shown to exert a strong pro-oxidant action on aerobic yeast cells. In addition these acids are mutagenic toward the yeast mitochondrial genome, even at levels that are subinhibitory to growth. This raises the concern that the large-scale consumption of these preservatives in the human diet may generate oxidative stress within the epithelia of the gastrointestinal tract.

The Requirement for Yeast Superoxide Dismutase Is Bypassed through Mutations in BSD2, a Novel Metal Homeostasis Gene

Oxygen toxicity in Saccharomyces cerevisiae strains lacking superoxide dismutase can be suppressed through mutations in either the BSD1 or BSD2 gene. In this report, we demonstrate that the BSD2 gene normally functions in the homeostasis of heavy metal ions. A mutation in BSD2 not only reverses the aerobic defects of yeast strains lacking superoxide dismutase but also is associated with an increased sensitivity to copper and cadmium toxicity and an elevation in copper ion accumulation. The BSD2 gene was cloned by functional complementation and is predicted to encode a novel 37.5-kDa protein with three potential transmembrane domains. The mutant bsd2-1 allele was isolated and found to contain a single C-to-T transition changing a centrally located proline to a serine. This substitution results in total inactivation of BSD2, since the bsd2-1 mutation is identical to a bsd2 delta gene deletion in phenotype. BSD2 is expressed in yeast cells as a 1.5-kb mRNA. Although the gene functions in copper detoxification, BSD2 is not induced by copper ions, as is the case with S. cerevisiae metallothioneins. A probable role for copper ions in the bsd2 reversal of oxidative damage is discussed.

The requirement for yeast superoxide dismutase is bypassed through mutations in BSD2, a novel metal homeostasis gene.

Oxygen toxicity in Saccharomyces cerevisiae strains lacking superoxide dismutase can be suppressed through mutations in either the BSD1 or BSD2 gene. In this report, we demonstrate that the BSD2 gene normally functions in the homeostasis of heavy metal ions. A mutation in BSD2 not only reverses the aerobic defects of yeast strains lacking superoxide dismutase but also is associated with an increased sensitivity to copper and cadmium toxicity and an elevation in copper ion accumulation. The BSD2 gene was cloned by functional complementation and is predicted to encode a novel 37.5-kDa protein with three potential transmembrane domains. The mutant bsd2-1 allele was isolated and found to contain a single C-to-T transition changing a centrally located proline to a serine. This substitution results in total inactivation of BSD2, since the bsd2-1 mutation is identical to a bsd2 delta gene deletion in phenotype. BSD2 is expressed in yeast cells as a 1.5-kb mRNA. Although the gene functions in copper detoxification, BSD2 is not induced by copper ions, as is the case with S. cerevisiae metallothioneins. A probable role for copper ions in the bsd2 reversal of oxidative damage is discussed.

Screening of thermotolerant yeasts as producers of superoxide dismutase.

A screening procedure for highly thermostable yeast superoxide dismutase was developed. Growth yields at various temperatures were estimated for ten mesophilic and thermotolerant strains, belonging to the genera Saccharomyces, Kluyveromyces and Pichia. Higher yields at 45 degrees C were obtained for K. lactis 90-3 and 90-4. A correlation between the ability to grow at higher temperature and the thermostability of the superoxide dismutase enzyme synthesized was observed. A comparison of the operational stability of the superoxide dismutase of all tested strains suggests that the enzyme of K. lactis strains was more thermostable than that of the other tested microorganisms.

Screening of thermotolerant yeasts as producers of superoxide dismutase

A screening procedure for highly thermostable yeast superoxide dismutase was developed. Growth yields at various temperatures were estimated for ten mesophilic and thermotolerant strains, belonging to the genera Saccharomyces, Kluyveromyces and Pichia. Higher yields at 45 degrees C were obtained for K. lactis 90-3 and 90-4. A correlation between the ability to grow at higher temperature and the thermostability of the superoxide dismutase enzyme synthesized was observed. A comparison of the operational stability of the superoxide dismutase of all tested strains suggests that the enzyme of K. lactis strains was more thermostable than that of the other tested microorganisms.

Crystal structure of yeast Cu,Zn superoxide dismutase: Crystallographic refinement at 2.5 Å resolution

The structure of Cu,Zn yeast superoxide dismutase has been determined to 2.5 Å resolution. The enzyme crystallizes in the P2 12 12 space group with two dimeric enzyme molecules per asymmetric unit. The structure has been solved by molecular replacement techniques using the dimer of the bovine enzyme as the search model, and refined by molecular dynamics with crystallographic pseudo-energy terms, followed by conventional crystallographic restrained refinement. The R-factor for 32,088 unique reflections in the 10.0 to 2.5 Å resolution range (98.2% of all possible reflections) is 0.158 for a model comprising two protein dimers and 516 bound solvent molecules, with a root-mean-square deviation of 0.016 Å from the ideal bond lengths, and an average B-factor value of 29.9 Å 2. A dimeric molecule of the enzyme is composed of two identical subunits related by a noncrystallographic 2-fold axis. Each subunit (153 amino acid residues) has as its structural scaffolding a flattened antiparallel eight-stranded β-barrel, plus three external loops. The overall three-dimensional structure is quite similar to the phylogenetically distant bovine superoxide dismutase (55% amino acid homology), the largest deviations can be observed in the regions of amino acid insertions. The major insertion site hosting residues Ser25A and Gly25B, occurs in the 2,3 β-turn between strands 2b and 3c, resulting in the structural perturbations of the two neighbouring strands. The second insertion site, at the end of the 3c β-strand in the wide Greek-key loop, hosts the Asn35A residue, having an evident effect on the structure of the loop and possibly on the neighbouring 5,4 β-turn. The salt bridge Arg77-Asp99 and the disulphide bridge Cys55-Cys144 stabilize the loop regions containing the metal ligands. The stereochemistry of the two metal centres is conserved, with respect to the bovine enzyme. The Cu 2+ ligands show an uneven distortion from a square plane, while Zn 2+ co-ordination geometry is distorted tetrahedral. The imidazole ring of the His61 residue forms a bridge between Cu and Zn ions. A solvent peak compatible with a fifth ligand is observed 2.0 Å away from the copper in the active site channel, which is filled by ordered water molecules that possibly contribute to the stability and function of the enzyme. The charged residues responsible for the electrostatic guidance of the substrate to the active site (Glu130, Glu131, Lys134 and Arg141) are fairly conserved in their positions, some of them showing different interactions in the four chains due to the intermolecular contacts between the dimers. The β-barrel of the bovine enzyme is closed at opposite ends by apolar interactions involving Leu104 and Leu36; the latter is substituted by Ser36 in the yeast enzyme. Three concerted substitutions Ala87 → Thr, His41 → Arg and Glu119 → Ala allow for the formation of a “plug” for the β-barrel by means of a hydrogen bonding scheme.

Structure solution and molecular dynamics refinement of the yeast Cu,Zn enzyme superoxide dismutase

Cu,Zn yeast superoxide dismutase was crystallized from polyethylene glycol solutions. The crystals belong to the P2(1)2(1)2 space group, with cell dimensions a = 105.3, b = 143.0, c = 62.1 A; two dimers of Mr = 32,000 each are contained in the asymmetric unit. Diffraction data at 2.5 A resolution were collected with the image-plate system at the EMBL synchrotron radiation facility in Hamburg. The structure was determined by molecular replacement using as a search model the 'blue-green' dimer of the bovine Cu,Zn superoxide dismutase. The crystallographic refinement of the molecular replacement solution was performed by means of molecular dynamics techniques and resulted in an R factor of 0.268 for the data between 6.0 and 2.5 A. The model was subsequently subjected to conventional restrained crystallographic refinement of the coordinates and temperature factors. The current R value for the data between 6.0 and 2.5 A is 0.220. Owing to the large radius of convergence of the molecular dynamics-crystallographic refinement, the convergence of the refinement process was reached after 18.1 ps of simulation time. The geometry of the active site of the enzyme appears essentially preserved compared with the bovine superoxide dismutase. The beta-barrel structure in the yeast enzyme is closed at the upper part by an efficient hydrogen-bonding scheme.

Binding of fluoride to copper zinc superoxide dismutase

The interaction of fluoride with copper zinc superoxide dismutase (SOD) has been reinvestigated by using both bovine and yeast isoenzymes. The affinity of anions for the latter isoenzyme is larger, and this avoids much of the ionic strength effects connected with weak binding ligands. 19 F NMR studies on fluoride in presence of yeast SOD confirm that the anion binds the copper ion. 1 H NMR studies of the Cu 2 Co 2 SOD derivatives in the presence of F − indicate that no ligand is removed from coordination

Purification and characterization of an N alpha-acetyltransferase from Saccharomyces cerevisiae.

N alpha-Acetyltransferase, which catalyzes the transfer of an acetyl group from acetyl coenzyme A to the alpha-NH2 group of proteins and peptides, was isolated from Saccharomyces cerevisiae and demonstrated by protein sequence analysis to be NH2-terminally blocked. The enzyme was purified 4,600-fold to apparent homogeneity by successive purification steps using DEAE-Sepharose, hydroxylapatite, DE52 cellulose, and Affi-Gel blue. The Mr of the native enzyme was estimated to be 180,000 +/- 10,000 by gel filtration chromatography, and the Mr of each subunit was estimated to be 95,000 +/- 2,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has a pH optimum near 9.0, and its pI is 4.3 as determined by chromatofocusing on Mono-P. The enzyme catalyzed the transfer of an acetyl group to various synthetic peptides, including human adrenocorticotropic hormone (ACTH) (1-24) and its [Phe2] analogue, yeast alcohol dehydrogenase I (1-24), yeast alcohol dehydrogenase II (1-24), and human superoxide dismutase (1-24). These peptides contain either Ser or Ala as NH2-terminal residues which together with Met are the most commonly acetylated NH2-terminal residues (Persson, B., Flinta, C., von Heijne, G., and Jornvall, H. (1985) Eur. J. Biochem. 152, 523-527). Yeast enolase, containing a free NH2-terminal Ala residue, is known not to be N alpha-acetylated in vivo (Chin, C. C. Q., Brewer, J. M., and Wold, F. (1981) J. Biol. Chem. 256, 1377-1384), and enolase (1-24), a synthetic peptide mimicking the protein's NH2 terminus, was not acetylated in vitro by yeast acetyltransferase. The enzyme did not catalyze the N alpha-acetylation of other synthetic peptides including ACTH(11-24), ACTH(7-38), ACTH(18-39), human beta-endorphin, yeast superoxide dismutase (1-24). Each of these peptides has an NH2-terminal residue which is rarely acetylated in proteins (Lys, Phe, Arg, Tyr, Val, respectively). Among a series of divalent cations, Cu2+ and Zn2+ were demonstrated to be the most potent inhibitors. The enzyme was inactivated by chemical modification with diethyl pyrocarbonate and N-bromosuccinimide.

Structural analyses of various Cu 2+, Zn 2+-superoxide dismutases by differential scanning calorimetry and Raman spectroscopy

The thermal denaturation profile of the Cu 2+, Zn 2+ metalloenzyme, bovine Superoxide dismutase, consists of two primary components, the major component denatures irreversibly at T m = 104 °C with a total enthalpy (Δ H cal) of 7.30 cal/g. Reduction of Cu(II) to Cu(I) with potassium ferrocyanide lowers T m to 96 °C and Δ H cal to 6.96 cal/ g. The apo-form of bovine superoxide dismutase (both Cu and Zn removed) denatures at 60 °C with an enthalpy only one-half that of the holo-form. The reduced thermal stability, which indicates a greater ability to change conformation, may explain the previously observed much greater membrane binding of the apo-enzyme. Reconstitution with Zn 2+, Cu 2+, or Zn 2+ and Cu 2+ raises T m to 80, 89, or 102 °C, respectively, with corresponding increases in the enthalpy. Thus, the metal ions considerably stabilize the enzyme and must somewhat affect conformation. The effect of Cu 2+ alone is greater than that of Zn 2+, although both are needed for full stability. Raman spectroscopy indicates little difference in secondary structure between the apo- and holo-forms, implying that the increased stability due to metal binding is not caused by an extreme structural reorganization. The value of T m of canine and yeast superoxide dismutase is also lowered by reduction of Cu(II). The reduced form of the yeast enzyme denatures irreversibly, as do all forms of the bovine and canine enzymes, but the oxidized form is unique in that it denatures reversibly. Thus, the copper ion must be oxidized for renaturation and appears to act as a nucleation site.

A proposal for the metal geometry in yeast superoxide dismutase based on results from EXAFS spectroscopy

Extended x-ray absorption fine structure (EXAFS) spectra have been recorded at the Cu edge and Zn edge in native yeast superoxide dismutase and at the Cu edge and Cd edge in the yeast superoxide dismutase derivative, where Zn has been substituted with Cd. Two different metal ligand distances in the range 1.9-2.0 A and 2.3-2.4 are determined for the Cu and Zn metals. For Cd in the Zn site two different metal ligand distances about 2.2 A and 2.6 A, respectively, were found. The striking feature is the similarity between the amplitude and radii determined for both the Cu and Zn sites. The increased distances for Cd can be explained by the increased ionic radius of Cd relative to Cu and Zn. Based on these EXAFS results and other relevant knowledge about the metal geometries, we propose that histidine 61 (63) positioned between the Cu and Zn metals are in one subunit bound to Zn and in the other to Cu. This model explains the recently observed difference between the two metal sites in each subunit.

Affinity inactivation of bovine Cu, Zn superoxide dismutase by hydroperoxide anion, HO 2−

Bovine liver Cu,Zn superoxide dismutase (SOD) is inactivated by hydrogen peroxide at alkaline pH, and full inactivation correlates with the loss of 1.1 histidine/subunit. At each pH utilized, saturation of the rate of inactivation is observed. This process is characterized by a half-saturation constant for peroxide and a maximum pseudo-first-order rate constant for inactivation. At 25 degrees C, the former decreases from 15.7 to 3.2 mM as the pH is increased from 9.0 to 11.5, while the latter increases from 0.83 to 2.43 per min over the same pH range. We have previously (Arch. Biochem. Biophys. 224, 579 (1983] proposed that the true affinity reagent for the inactivation of yeast SOD is the hydroperoxide anion, and we now believe the same is true for bovine SOD. However, a subtle difference between the two enzymes exists, for while the maximum pseudo-first-order rate constant for inactivation of bovine SOD increases with increasing pH, the same parameter for the yeast enzyme is pH-independent.

Reversibility of the thermal denaturation of yeast superoxide dismutase

Reversibility of the thermal denaturation of yeast superoxide dismutase

Interaction of superoxide dismutase with phospholipid liposomes an uptake, spin label and calorimetric study

The effect of superoxide dismutases from five species upon phospholipid bilayers has been investigated. The uptake by egg phosphatidylcholine bilayers of the holo and apo forms of bovine superoxide dismutase increases with enzyme concentration and only a fraction of each is removed by treatment with trypsin. These uptake data indicate that both forms of the enzyme associate with and are embedded within lipid bilayers. From the spectrum of the spin label 2-(3-carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinyloxyl, the binding of superoxide dismutase to egg phosphatidylcholine bilayers can be shown to disorder the lipid packing. The disordering by the bovine holoenzyme is small but increases with increasing enzyme concentration and period of incubation. The disordering effects of the apoenzyme are much larger and are reversible by Cu 2+, Zn 2+ reconstitution of the apoenzyme. The disordering effect of the apoenzyme is further confirmed by differential scanning calorimetry. The gel to liquid crystalline phase transition of egg phosphatidylcholine is lowered 7°C by 25% by weight apo-superoxide dismutase to lipid. Human, dog, swordfish and yeast superoxide dismutases also disorder, and to a greater extent than the bovine enzyme. The greatest perturbation is produced by yeast superoxide dismutase; a 20% decrease in the order parameter by 50% by weight enzyme to lipid.