Screening of thermotolerant yeasts as producers of superoxide dismutase

Abstract 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°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.     

Role of tryptophan 161 in catalysis by human manganese superoxide dismutase.

Tryptophan 161 is a highly conserved residue that forms a hydrophobic side of the active site cavity of manganese superoxide dismutase (MnSOD), with its indole ring adjacent to and about 5 A from the manganese. We have made a mutant containing the conservative replacement Trp 161 --> Phe in human MnSOD (W161F MnSOD), determined its crystal structure, and measured the catalysis of the resulting mutant using pulse radiolysis to produce O(2)(*)(-). In the structure of W161F MnSOD the phenyl side chain of Phe 161 superimposes on the indole ring of Trp 161 in the wild type. However, in the mutant, the hydroxyl side chain of Tyr 34 is 3.9 A from the manganese, closer by 1.2 A than in the wild type. The tryptophan in MnSOD is not essential for the half-cycle of catalytic activity involving reduction of the manganese; the mutant W161F MnSOD had k(cat)/K(m) at 2.5 x 10(8) M(-)(1) s(-)(1), reduced only 3-fold compared with wild type. However, this mutant exhibited a strong product inhibition with a zero-order region of superoxide decay slower by 10-fold compared with wild type. The visible absorption spectrum of W161F MnSOD in the inhibited state was very similar to that observed for the inhibited wild-type enzyme. The appearance of the inhibited form required reaction of 2 molar equiv of O(2)(*)(-) with W161F Mn(III)SOD, one to form the reduced state of the metal and the second to form the inhibited complex, confirming that the inhibited complex requires reaction of O(2)(*)(-) with the reduced form of the enzyme. This work suggests that a significant role of Trp 161 in the active site is to promote the dissociation of product peroxide, perhaps in part through its effect on the orientation of Tyr 34.     

Regulation by cytokines of extracellular superoxide dismutase and other superoxide dismutase isoenzymes in fibroblasts.

The influence of cytokines on extracellular superoxide dismutase (EC-SOD) expression by human dermal fibroblasts was investigated. The expression was markedly stimulated by interferon-gamma (IFN-gamma), was varying between fibroblast lines stimulated or depressed by interleukin-1 alpha (IL-1 alpha), was intermediately depressed by tumor necrosis factor-alpha (TNF-alpha), and markedly depressed by transforming growth factor-beta (TGF-beta). TNF-alpha, however, enhanced the stimulation by a high dose of IFN-gamma, whereas TGF-beta markedly depressed the stimulations given by IFN-gamma and IL-1 alpha. The ratio between the maximal stimulation and depression observed was around 30-fold. The responses were generally slow and developed over periods of several days. There were no effects of IFN-alpha, IL-2, IL-3, IL-4, IL-6, IL-8, granulocyte-macrophage colony-stimulating factor, human growth hormone, Escherichia coli lipopolysaccharide, leukotriene B4, prostaglandin E2, formylmethionylleucylphenylalanine, platelet-activating factor, and indomethacin. The cytokines influencing the EC-SOD expression are also known to influence superoxide production by leukocytes and other cell types, and the EC-SOD response pattern is roughly compatible with the notion that its function is to protect cells against extracellular superoxide radicals. The results show that EC-SOD is a participant in the complex inflammatory response orchestrated by cytokines. The CuZn-SOD activity of the fibroblasts was not influenced by any of the cytokines, whereas the Mn-SOD activity was depressed by TGF-beta. TNF-alpha, IL-1 alpha, and IFN-gamma stimulated the Mn-SOD activity, as previously known, and these responses were reduced by TGF-beta. The different responses of the three SOD isoenzymes illustrate their different physiological roles.     

Role of extracellular superoxide dismutase in bleomycin-induced pulmonary fibrosis

Bleomycin administration results in well-described intracellular oxidative stress that can lead to pulmonary fibrosis. The role of alveolar interstitial antioxidants in this model is unknown. Extracellular superoxide dismutase (EC-SOD) is the primary endogenous extracellular antioxidant enzyme and is abundant in the lung. We hypothesized that EC-SOD plays an important role in attenuating bleomycin-induced lung injury. Two weeks after intratracheal bleomycin administration, we found that wild-type mice induced a 106 +/- 25% increase in lung EC-SOD. Immunohistochemical staining revealed that a large increase in EC-SOD occurred in injured lung. Using mice that overexpress EC-SOD specifically in the lung, we found a 53 +/- 14% reduction in bleomycin-induced lung injury assessed histologically and a 17 +/- 6% reduction in lung collagen content 2 wk after bleomycin administration. We conclude that EC-SOD plays an important role in reducing the magnitude of lung injury from extracellular free radicals after bleomycin administration.     

Radioprotection by short-term oxidative preconditioning: Role of manganese superoxide dismutase

Manganese superoxide dismutase (MnSOD) is vital to the protection of mitochondria and cells against oxidative stress. Earlier, we demonstrated that catalytically active homo-tetramer of MnSOD can be stabilized by oxidative cross-linking. Here we report that this effect may be translated into increased radioresistance of mouse embryonic cells (MECs) by pre-exposure to oxidative stress. Pre-treatment of MECs with antimycin A, rotenone or H₂O₂ increased their survival after irradiation. Using MnSOD siRNA, we show that MECs with decreased MnSOD levels displayed a lowered ability to preconditioning. Thus oxidative preconditioning may be used for targeted regulation of MnSOD.

Structured summary

MINT-7288408: MnSOD (uniprotkb:P04179) and MnSOD (uniprotkb:P04179) physically interact (MI:0915) by zymography (MI:0512)     

Endocytosis of superoxide dismutase by rat liver.

We have investigated the endocytosis by rat liver of superoxide dismutase (SOD) labelled with 125I. (125I) SOD is quickly taken up by the liver where it remains in significant amounts for at least 150 min. Adsorptive endocytosis is probably involved. Distribution of radioactivity was established after differential and isopycnic centrifugation and compared with that of cathepsin C, a lysosomal enzyme. Results show that the behavior of radioactivity is similar to that of the hydrolase. SOD activity is only marginally affected by incubation in the presence of a purified lysosome extract; moreover, when (125I) SOD is treated in the same conditions, only a few percent of radioactivity becomes acidosoluble. These observations indicate that SOD taken up by the liver accumulates in lysosomes where it can stay for a relatively long time owing to its relative resistance to lysosomal hydrolases.     

Role of the electrostatic loop charged residues in Cu,Zn superoxide dismutase.

We have expressed and characterized a mutant of Xenopus laevis Cu,Zn superoxide dismutase in which four highly conserved charged residues belonging to the electrostatic loop have been replaced by neutral side chains: Lys120 --> Leu, Asp130 --> Gln, Glu131 --> Gln, and Lys134 --> Thr. At low ionic strength, the mutant enzyme is one of the fastest superoxide dismutases ever assayed (k = 6.7 x 10(9) M(-1) s(-1), at pH 7 and mu = 0.02 M). Brownian dynamics simulations give rise to identical enzyme-substrate association rates for both wild-type and mutant enzymes, ruling out the possibility that enhancement of the activity is due to pure electrostatic factors. Comparative analysis of the experimental catalytic rate of the quadruple and single mutants reveals the nonadditivity of the mutation effects, indicating that the hyperefficiency of the mutant is due to a decrease of the energy barrier and/or to an alternative pathway for the diffusion of superoxide within the active site channel. At physiological ionic strength the catalytic rate of the mutant at neutral pH is similar to that of the wild-type enzyme as it is to the catalytic rate pH dependence. Moreover, mutation effects are additive. These results show that, at physiological salt conditions, electrostatic loop charged residues do not influence the diffusion pathway of the substrate and, if concomitantly neutralized, are not essential for high catalytic efficiency of the enzyme, pointing out the role of the metal cluster and of the invariant Arg141 in determining the local electrostatic forces facilitating the diffusion of the substrate towards the active site.     

Peroxynitrite-mediated tyrosine nitration catalyzed by superoxide dismutase.

Peroxynitrite (ONOO-), the reaction product of superoxide (O2-) and nitric oxide (NO), may be a major cytotoxic agent produced during inflammation, sepsis, and ischemia/reperfusion. Bovine Cu,Zn superoxide dismutase reacted with peroxynitrite to form a stable yellow protein-bound adduct identified as nitrotyrosine. The uv-visible spectrum of the peroxynitrite-modified superoxide dismutase was highly pH dependent, exhibiting a peak at 438 nm at alkaline pH that shifts to 356 nm at acidic pH. An equivalent uv-visible spectrum was obtained by Cu,Zn superoxide dismutase treated with tetranitromethane. The Raman spectrum of authentic nitrotyrosine was contained in the spectrum of peroxynitrite-modified Cu,Zn superoxide dismutase. The reaction was specific for peroxynitrite because no significant amounts of nitrotyrosine were formed with nitric oxide (NO), nitrogen dioxide (NO2), nitrite (NO2-), or nitrate (NO3-). Removal of the copper from the Cu,Zn superoxide dismutase prevented formation of nitrotyrosine by peroxynitrite. The mechanism appears to involve peroxynitrite initially reacting with the active site copper to form an intermediate with the reactivity of nitronium ion (NO2+), which then nitrates tyrosine on a second molecule of superoxide dismutase. In the absence of exogenous phenolics, the rate of nitration of tyrosine followed second-order kinetics with respect to Cu,Zn superoxide dismutase concentration, proceeding at a rate of 1.0 +/- 0.1 M-1.s-1. Peroxynitrite-mediated nitration of tyrosine was also observed with the Mn and Fe superoxide dismutases as well as other copper-containing proteins.     

Role of superoxide dismutase in a copper-resistant strain of yeast

A copper-resistant yeast strain (R_Cu) which was cultured repeatedlyin a medium containing 1 mM Cu was found to contain a largeamount of superoxide dismutase. When yeast cells, which hadbeen grown under different conditions and accordingly had variouslevels of superoxide dismutase activity, were exposed to copperunder nongrowing conditions and plated onto normal medium, themore dismutase they had, the higher was their survival ratio.When the cells were exposed to cadmium instead of copper, thesurvival ratios were independent of their enzyme activity. Theresults suggest that the toxicity of a transition metal suchas copper is necessary to account for the toxicity of superoxideradicals produced by reactions of copper and thiols in the cellcomponents. On the other hand, when the same yeast cells wereplated directly onto copper-containing medium, only R_Cu showeda marked high survival ratio. Hence, it is concluded that thecells need to overcome the toxicity of superoxide radicals togrow in the copper-containing medium, but a more effective resistantmechanism(s) must be present in R_Cu cells. The role of superoxidedismutase induced in R_Cu is discussed. (Received May 2, 1980; )     

Biological protection by superoxide dismutase

Diol dehydrase from $\text{Aerobacter aerogenes}$ was dissociated into two different protein components or subunits, designated Components F and S, by chromatography on DEAE-cellulose. Neither component alone possessed any appreciable catalytic activity. Diol dehydrase activity was restored when the two components were combined. Both components were also required for inactivation of coenzyme B₁₂ by oxygen when incubation was carried out in the absence of substrate aerobically. The more acidic component, Component S, was a sulfhydryl protein sensitive to an alkylating agent, iodoacetamide. Coenzyme B₁₂ was not bound by the individual components, F or S, both of which were necessary for the cobamide binding. The presence of substrate, 1,2-propanediol, in eluting buffer retarded the dissociation of the enzyme.     

Extracellular superoxide dismutase and other superoxide dismutase isoenzymes in tissues from nine mammalian species.

The contents of extracellular superoxide dismutase, CuZn superoxide dismutase and Mn superoxide dismutase were determined in tissues from nine mammalian species. The pattern of CuZn superoxide dismutase distribution was similar in all species, with high activity in metabolically active organs such as liver and kidney and low activity in, for example, skeletal muscle. Mn superoxide dismutase activity was high in organs with high respiration, such as liver, kidney, and myocardium. Overall the Mn superoxide dismutase activity in organs was almost as high as the CuZn superoxide dismutase activity. The content of extracellular superoxide dismutase was, almost without exception, lower than the content of the other isoenzymes. The pattern of tissue distribution was distinctly different from those of CuZn superoxide dismutase and Mn superoxide dismutase. The tissue distribution of extracellular superoxide dismutase differed among species, but in general there was much in lungs and kidneys and little in skeletal muscle. In man, pig, sheep, cow, rabbit and mouse the overall tissue extracellular superoxide dismutase activities were similar to each other, whereas dog, cat and rat tissues contained distinctly less. There was no general correlation between the tissue extracellular superoxide dismutase activity of any of the various species and the variable plasma activity. The ratio between the plasma and the overall tissue activities was high, for some species over unity, providing further evidence for the notion that one role of extracellular superoxide dismutase is as a plasma protein.     

Polarographic determination of superoxide dismutase.

A polarographic procedure is described which allows determination of the catalytic constants for superoxide dismutase-catalyzed reactions. The method presents a single and rapid evaluation of the enzyme concentrations as well as determination of its activity under different conditions; e.g., pH between 9 and 13, presence of urea, guanidine, sodium dodecyl sulphate and inhibitors such as CN^? and N₃^?.The results fit very well with data previously obtained with other methods and show that this polarographic procedure can be used under conditions that render the other methods unsuitable for the measurement of the enzyme activity.     

Role of superoxide dismutase in defense against SO2 toxicity and an increase in superoxide dismutase activity with SO2 fumigation

The role of superoxide dismutase (SOD) in defense against SO₂toxicity was investigated using leaves of poplar and spinach.Young poplar leaves having five times the SOD of the old leaveswere more resistant to the toxicity of SO₂. Spraying spinachleaves with diethyldithiocarbamate caused a marked loss of SODactivity which resulted in a decrease in their resistance tothe toxic effects of SO₂. The SOD activity in poplar leaveswas increased by fumigation with 0.1 ppm SO₂, and this was moreevident in young leaves than in old ones. The increased SODactivity was inhibited by cyanide. The poplar leaves havinghigh SOD activity induced with SO₂ fumigation were more resistantto 2.0 ppm SO₂ than the control leaves. These findings suggestthat SO₂ toxicity is in part due to the superoxide radical andthat SOD participates in the defense mechanism against SO₂ toxicity. (Received February 12, 1980; )     

In vivo inhibition of superoxide dismutase in mice by diethyldithiocarbamate.

Superoxide dismutase was assayed by a method which takes advantage of the inhibitory action of superoxide dismutase (or tissues which contain superoxide dismutase) on the rate of autooxidation of 6-hydroxydopamine. Incubation of pure superoxide dismutase of homogenates of brain or liver with 10(-3) M diethyldithiocarbamate for 1.5 hours resulted in total loss of superoxide dismutase activity. Inhibition of superoxide dismutase was not reversed by dialysis, but after dialysis, enzymatic activity was restored with CuSO4. When 1.5 g of diethyldithiocarbamate/kg were injected into mice, the superoxide dismutase activity at 3 hours was decreased by 86%, 71%, and 48%, respectively, in whole blood, liver, and brain. A dose of 0.5 g of diethyldithiocarbamate/kg lowered the superoxide dismutase activity by 42% in liver at 3 hours. A study of the time course for inhibiton of superoxide dismutase in liver after 1.5 g of diethyldithiocarbamate/kg, showed a maximum decrease (81%) within 1 hour, with a slow return to 64% of normal by 24 hours. Inhibition of superoxide dismutase in vivo and in vitro was confirmed with other assay systems based on the autooxidation of pyrogallol or epinephrine or on reduction of cytochrome c or intro blue tetrazolium. Treatment of animals with diethyldithiocarbamate may provide a useful experimental model to study the role of superoxide dismutase in various tissues.     

Role of hydrogen bonding in the active site of human manganese superoxide dismutase

The side chain of Gln143, a conserved residue in manganese superoxide dismutase (MnSOD), forms a hydrogen bond with the manganese-bound solvent and is critical in maintaining catalytic activity. The side chains of Tyr34 and Trp123 form hydrogen bonds with the carboxamide of Gln143. We have replaced Tyr34 and Trp123 with Phe in single and double mutants of human MnSOD and measured their catalytic activity by stopped-flow spectrophotometry and pulse radiolysis. The replacements of these side chains inhibited steps in the catalysis as much as 50-fold; in addition, they altered the gating between catalysis and formation of a peroxide complex to yield a more product-inhibited enzyme. The replacement of both Tyr34 and Trp123 in a double mutant showed that these two residues interact cooperatively in maintaining catalytic activity. The crystal structure of Y34F/W123F human MnSOD at 1.95 A resolution suggests that this effect is not related to a conformational change in the side chain of Gln143, which does not change orientation in Y34F/W123F, but rather to more subtle electronic effects due to the loss of hydrogen bonding to the carboxamide side chain of Gln143. Wild-type MnSOD containing Trp123 and Tyr34 has approximately the same thermal stability compared with mutants containing Phe at these positions, suggesting the hydrogen bonds formed by these residues have functional rather than structural roles.     

Role of the electrostatic loop of Cu,Zn superoxide dismutase in the copper uptake process.

Cu,Zn superoxide dismutases are characterized by the presence of four highly conserved charged residues (Lys120, Glu/Asp130, Glu131 and Lys134), which are placed at the edge of the active site channel and have been shown to be individually involved in the electrostatic attraction of the substrate toward the catalytically active copper ion. By genetic engineering we mutated these four residues into neutrally charged ones (Leu120, Gln130, Gln131, Thr134). The effects of these mutations on the rate of superoxide dismutation were not dramatic. In fact, at two different pH and ionic strength values, the mutant enzyme had a catalytic constant even higher with respect to the wild-type protein, showing that electrostatic interaction at these surface sites is not essential for high catalytic efficiency of the enzyme. The mutant and the wild-type enzyme showed the same degree of inhibition by CN(-), and both were not affected by I(-), showing that mutations did not alter the sensitivity of the enzyme to anions. On the other hand, reconstitution of active enzyme from either the wild-type or mutant copper-free enzymes with a copper(I)-glutathione [Cu(I)-GSH] complex showed that metal uptake by the mutant was much slower than by the wild-type enzyme. The demonstration that the 'electrostatic loop' is apparently conserved to assure optimal copper uptake by the enzyme, rather than fast dismutation, may provide further support to the idea that Cu,Zn superoxide dismutase is a bifunctional protein, acting in cellular defense against oxidative stress both as a copper buffer and as a superoxide radical scavenger.     

Metal uptake by manganese superoxide dismutase

Manganese superoxide dismutase is an important antioxidant defense metalloenzyme that protects cells from damage by the toxic oxygen metabolite, superoxide free radical, formed as an unavoidable by-product of aerobic metabolism. Many years of research have gone into understanding how the metal cofactor interacts with small molecules in its catalytic role. In contrast, very little is presently known about how the protein acquires its metal cofactor, an important step in the maturation of the protein and one that is absolutely required for its biological function. Recent work is beginning to provide insight into the mechanisms of metal delivery to manganese superoxide dismutase in vivo and in vitro.     

Elevation of superoxide dismutase in Halobacterium halobium by heat shock.

Exposure of Halobacterium halobium to 50 degrees C for 2.5 h in an aerobic environment resulted in a greater than twofold increase in the activity of the manganese-containing superoxide dismutase. Nondenaturing polyacrylamide gels stained for enzymatic activity did not reveal any additional isozymes of superoxide dismutase induced by the heat shock. The maximal effect was observed at 50 degrees C, and the elevated levels of activity remained constant during 5 h of recovery at 40 degrees C. The induction of enzymatic activity was sensitive to protein synthesis inhibitors. The results are discussed relative to heat shock and stress-related proteins as well as alterations in metabolism brought about by elevated temperatures.