Marker-free chromosomal integration of the manganese superoxide dismutase gene (sodA) from Streptococcus thermophilus into Lactobacillus gasseri

A strategy for functional gene replacement in the chromosome of Lactobacillus gasseri is described. The phospho-beta-galactosidase II gene (lacII) was functionally replaced by the manganese superoxide dismutase (MnSOD) gene (sodA) from Streptococcus thermophilus, by adapting the insertional inactivation method described for lactobacilli [Russell, W.M. and Klaenhammer, T.R. 2001 Efficient system for directed integration into the Lactobacillus acidophilus and Lactobacillus gasseri chromosomes via homologous recombination. Appl. Environ. Microbiol. 67, 4361-4364]. L. gasseri carrying the heterologous sodA gene grew on lactose as efficiently as the wild-type parent. An active MnSOD was expressed in the transgenic strain, and the enzyme migrated on PAGE-SOD activity gels to the same position as that of MnSOD from S. thermophilus. The expression of MnSOD from a single copy of sodA integrated in the chromosome of L. gasseri provided enhanced tolerance to hydrogen peroxide, and extended the viability of carbon/energy starved cultures stored at 25 degrees C. This is the first report showing the successful utilization of the pORI plasmids system to generate marker-free gene integration in L. gasseri strains.     

In vitro preparation of iron-substituted human manganese superoxide dismutase: possible toxic properties for mitochondria

We prepared an iron-substituted form of recombinant human manganese superoxide dismutase (MnSOD) by using guanidine hydrochloride for the first time as a model of iron-misincorporated MnSOD, the formation of which has been reported by M. Yang et al. upon disruption of mitochondrial metal homeostasis in yeast (Yang et al. 2006, EMBO J. 25, 1775-1783). The iron-substituted enzyme contained 0.79 g atoms of Fe/mol of subunits and had a specific activity of 80 units/mg protein/g atom of Fe/mol of subunit, which was less than 3% of the activity of the purified MnSOD. Fe-substituted MnSOD (Fe-MnSOD) showed the same absorption spectrum as that of bacterial Fe-MnSODs reported, a similar pH-dependent change of the enzymatic activity, and a similar electron paramagnetic resonance spectrum. Fe-MnSOD showed more thermal stability than native MnSOD. The Fe-substituted enzyme showed a hydrogen-peroxide-mediated radical-generating activity, which was monitored by a cation radical of 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonate) formation similar to that of Cu,ZnSOD, but native human MnSOD and FeSOD showed no radical-generation ability. This evidence suggests that a substitution of Mn to Fe in human MnSOD in mitochondria may produce a disadvantage for oxidative stress in three ways: loss of the enzymatic activity, increase of stability, and gain of radical-generating ability.     

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.     

Structural mobility in human manganese superoxide dismutase

Human manganese superoxide dismutase (MnSOD) is a homotetramer of 22 kDa subunits, a dimer of dimers containing dimeric and tetrameric interfaces. We have investigated conformational mobility at these interfaces by measuring amide hydrogen/deuterium (H/D) exchange kinetics and 19F NMR spectra, both being excellent methods for analyzing local environments. Human MnSOD was prepared in which all nine tyrosine residues in each subunit are replaced with 3-fluorotyrosine. The 19F NMR spectrum of this enzyme showed five sharp resonances that have been assigned by site-specific mutagenesis by replacing each 3-fluorotyrosine with phenylalanine; four 19F resonances not observed are near the paramagnetic manganese and extensively broadened. The temperature dependence of the line widths and chemical shifts of the 19F resonances were used to estimate conformational mobility. 3-Fluorotyrosine 169 at the dimeric interface showed little conformational mobility and 3-fluorotyrosine 45 at the tetrameric interface showed much greater mobility by these measures. In complementary studies, H/D exchange mass spectrometry was used to measure backbone dynamics in human MnSOD. Using this approach, amide hydrogen exchange kinetics were measured for regions comprising 78% of the MnSOD backbone. Peptides containing Tyr45 at the tetrameric interface displayed rapid exchange of hydrogen with deuterium while peptides containing Tyr169 in the dimeric interface only displayed moderate exchange. Taken together, these studies show that residues at the dimeric interface, such as Tyr169, have significantly less conformational freedom or mobility than do residues at the tetrameric interface, such as Tyr45. This is discussed in terms of the role in catalysis of residues at the dimeric interface.     

Induction of superoxide dismutase and cytotoxicity by manganese in human breast cancer cells.

MnCl2 induced manganese-containing superoxide dismutase (MnSOD) expression (mRNA, immunoreactive protein, and enzyme activity) in human breast cancer Hs578T cells. The induction of MnSOD immunoreactive protein in Hs578T cells was inhibited by tiron (a metal chelator and superoxide scavenger), pyruvate (a hydrogen peroxide scavenger), or 2-deoxy-d-glucose (DG, an inhibitor of glycolysis and the hexose monophosphate shunt), but not by 5,5-dimethyl-1-pyrroline-1-oxide (a superoxide scavenger), N-acetyl cysteine (a scavenger for reactive oxygen species and precursor of glutathione), diphenylene iodonium (an inhibitor of flavoproteins such as NADPH oxidase and nitric oxide synthase), or SOD (a superoxide scavenger). Northern blotting demonstrated that tiron or DG affected at the mRNA level, while pyruvate affected Mn-induced MnSOD expression at both the mRNA and protein levels. These results demonstrate that Mn can induce MnSOD expression in cultured human breast cancer cells. Mn also induced apoptosis and necrosis in these cells. Since inhibitors of Mn-induced MnSOD induction did not affect cell viability, MnSOD induction is probably not the cause of the Mn-induced cell killing.     

Characterization of superoxide dismutase in Streptococcus thermophilus.

Streptococcus thermophilus AO54 possesses a single manganese-containing superoxide dismutase (MnSOD). The enzyme was found to be insensitive to cyanide or to a modified H2O2 treatment. The enzyme is expressed in a growth-phase-dependent fashion, increasing three- to fourfold upon entry into stationary phase. The specific activity for MnSOD was the same under anaerobic or aerobic conditions and was not induced by the presence of paraquat under aerobic conditions.     

Role of a glutamate bridge spanning the dimeric interface of human manganese superoxide dismutase

The function in the structure, stability, and catalysis of the interfaces between subunits in manganese superoxide dismutase (MnSOD) is currently under scrutiny. Glu162 in homotetrameric human MnSOD spans a dimeric interface and forms a hydrogen bond with His163 of an adjacent subunit which is a direct ligand of the manganese. We have examined the properties of two site-specific mutants of human MnSOD in which Glu162 is replaced with Asp (E162D) and Ala (E162A). The X-ray crystal structures of E162D and E162A MnSOD reveal no significant structural changes compared with the wild type other than the removal of the hydrogen bond interaction with His163 in E162A MnSOD. In the case of E162D MnSOD, an intervening solvent molecule fills the void created by the mutation to conserve the hydrogen bond interaction between His163 and residue 162. These mutants retain their tetrameric structure and their specificity for manganese over iron. Each has catalytic activity in the disproportionation of superoxide that is typically 5-25% of that of the wild-type enzyme and a level of product inhibition greater by approximately 2-fold. Differential scanning calorimetry indicates that the hydrogen bond between Glu162 and His163 contributes to the stability of MnSOD, with the major unfolding transition occurring at 81 degrees C for E162A compared to 90 degrees C for wild-type MnSOD. These results suggest that Glu162 at the tetrameric interface in human MnSOD supports stability and efficient catalysis and has a significant role in regulating product inhibition.     

Structural and kinetic study of differences between human and Escherichia coli manganese superoxide dismutases

Human manganese superoxide dismutase (MnSOD) is characterized by a product inhibition stronger than that observed in bacterial forms of MnSOD. Previous studies show that the conserved, active-site residue Tyr34 mediates product inhibition; however, the protein environment of Tyr34 is different in human and Escherichia coli MnSOD. We have prepared two site-specific mutants of human MnSOD with replacements of Phe66 with Ala and Leu (F66A and F66L, respectively), altering the surroundings of Tyr34. Pulse radiolysis was used to generate superoxide, and measurements of catalysis were taken in single-turnover experiments by observing the visible absorbance of species of MnSOD and under catalytic conditions observing the absorbance of superoxide. The mutation of Phe66 to Leu resulted in a mutant of human MnSOD with weakened product inhibition resembling that of E. coli MnSOD. Moreover, the mechanism of this weakened product inhibition was similar to that in E. coli MnSOD, specifically a decrease in the rate constant for the oxidative addition of superoxide to Mn2+MnSOD leading to the formation of the peroxide-inhibited enzyme. In addition, the crystal structures of both mutants have been determined and compared to those of wild-type human and E. coli MnSOD. The crystallographic data suggest that the solvent structure and its mobility as well as side chain conformations may affect the extent of product inhibition. These data emphasize the role of residue 66 in catalysis and inhibition and provide a structural explanation for differences in catalytic properties between human and certain bacterial forms of MnSOD.     

Expression of a heterologous manganese superoxide dismutase gene in intestinal lactobacilli provides protection against hydrogen peroxide toxicity

In living organisms, exposure to oxygen provokes oxidative stress. A widespread mechanism for protection against oxidative stress is provided by the antioxidant enzymes: superoxide dismutases (SODs) and hydroperoxidases. Generally, these enzymes are not present in Lactobacillus spp. In this study, we examined the potential advantages of providing a heterologous SOD to some of the intestinal lactobacilli. Thus, the gene encoding the manganese-containing SOD (sodA) was cloned from Streptococcus thermophilus AO54 and expressed in four intestinal lactobacilli. A 1.2-kb PCR product containing the sodA gene was cloned into the shuttle vector pTRK563, to yield pSodA, which was functionally expressed and complemented an Escherichia coli strain deficient in Mn and FeSODs. The plasmid, pSodA, was subsequently introduced and expressed in Lactobacillus gasseri NCK334, Lactobacillus johnsonii NCK89, Lactobacillus acidophilus NCK56, and Lactobacillus reuteri NCK932. Molecular and biochemical analyses confirmed the presence of the gene (sodA) and the expression of an active gene product (MnSOD) in these strains of lactobacilli. The specific activities of MnSOD were 6.7, 3.8, 5.8, and 60.7 U/mg of protein for L. gasseri, L. johnsonii, L. acidophilus, and L. reuteri, respectively. The expression of S. thermophilus MnSOD in L. gasseri and L. acidophilus provided protection against hydrogen peroxide stress. The data show that MnSOD protects cells against hydrogen peroxide by removing O(2)(.-) and preventing the redox cycling of iron. To our best knowledge, this is the first report of a sodA from S. thermophilus being expressed in other lactic acid bacteria.     

Overexpression of manganese or copper-zinc superoxide dismutase inhibits breast cancer growth

We have studied the effects of overexpression of superoxide dismutase (SOD), a tumor suppressor protein that dismutes superoxide radical to H2O2, on breast cancer cell growth in vitro and xenograft growth in vivo. No previous work has directly compared the growth-suppressive effects of manganese SOD (MnSOD) and copper-zinc SOD (CuZnSOD). We hypothesized that either adenoviral MnSOD (AdMnSOD) or adenoviral CuZnSOD (AdCuZnSOD) gene therapy would suppress the growth of human breast cancer cells. After determining the antioxidant profiles of three human breast cell lines, MCF 10A, MDA-MB231, and MCF-7, we measured the effects of MnSOD or CuZnSOD overexpression on cell growth and survival in vitro and in vivo. Results demonstrated that infection with AdMnSOD or AdCuZnSOD increased the activity of the respective enzyme in all three cell lines. In vitro, overexpression of MnSOD or CuZnSOD decreased not only cell growth but also clonogenic survival in a dose- and transgene-dependent manner. In vivo, treatment of tumors with AdMnSOD or AdCuZnSOD decreased xenograft growth compared to controls. The first direct comparison of MnSOD to CuZnSOD overexpression indicated that CuZnSOD and MnSOD were similarly effective at suppressing cancer cell growth.     

Redox properties of human manganese superoxide dismutase and active-site mutants

The redox potential of human manganese superoxide dismutase (MnSOD) has been difficult to determine because of the problem of finding suitable electron mediators. We have found that ferricyanide and pentacyanoaminoferrate can be used as electron mediators, although equilibration is very slow with a half-time near 6 h. Values of the midpoint potential were determined both by allowing enzyme and mediators to equilibrate up to 38 h and by reductive titration adding dithionite to enzyme and mediator. An overall value of the midpoint potential was found to be 393 +/- 29 mV. To elucidate the role of His30 and Tyr34 in the active site of human MnSOD, we have also measured the redox properties of the site-specific mutants His30Asn (H30N) and Tyr34Phe (Y34F) and compared them with the wild-type enzyme. Crystal structures have shown that each mutation interrupts a hydrogen bond network in the active site, and each causes a 10-fold decrease in the maximal velocity of catalysis of superoxide dismutation as compared with wild type. The present study shows that H30N and Y34F human MnSOD have very little effect, within experimental uncertainty, on the redox potential of the active-site metal. The redox potentials determined electrochemically were 365 +/- 28 mV for H30N and 435 +/- 30 mV for Y34F MnSOD. These results suggest that the role of His30 and Tyr34 is more in support of catalysis, probably proton transport, and not in the tuning of the redox potential.     

Multiple replacements of glutamine 143 in human manganese superoxide dismutase: effects on structure, stability, and catalysis

Glutamine 143 in human manganese superoxide dismutase (MnSOD) forms a hydrogen bond with the manganese-bound solvent molecule and is investigated by replacement using site-specific mutagenesis. Crystal structures showed that the replacement of Gln 143 with Ala made no significant change in the overall structure of the mutant enzyme. Two new water molecules in Q143A MnSOD were situated in positions nearly identical with the Oepsilon1 and Nepsilon2 of the replaced Gln 143 side chain and maintained a hydrogen-bonded network connecting the manganese-bound solvent molecule to other residues in the active site. However, their presence could not sustain the stability and activity of the enzyme; the main unfolding transition of Q143A was decreased 16 degrees C and its catalysis decreased 250-fold to k(cat)/K(m) = 3 x 10(6) M(-)(1) s(-)(1), as determined by stopped-flow spectrophotometry and pulse radiolysis. The mutant Q143A MnSOD and other mutants at position 143 showed very low levels of product inhibition and favored Mn(II)SOD in the resting state, whereas the wild type showed strong product inhibition and favored Mn(III)SOD. However, these differences did not affect the rate constant for dissociation of the product-inhibited complex in Q143A MnSOD which was determined from a characteristic absorbance at 420 nm and was comparable in magnitude ( approximately 100 s(-)(1)) to that of the wild-type enzyme. Hence, Gln 143, which is necessary for maximal activity in superoxide dismutation, appears to have no role in stabilization and dissociation of the product-inhibited complex.     

Enhanced drought tolerance of transgenic rice plants expressing a pea manganese superoxide dismutase

We investigated the role that manganese superoxide dismutase (MnSOD), an important antioxidant enzyme, may play in the drought tolerance of rice. MnSOD from pea (Pisum sativum) under the control of an oxidative stress-inducible SWPA2 promoter was introduced into chloroplasts of rice (Oryza sativa) by Agrobacterium-mediated transformation to develop drought-tolerant rice plants. Functional expression of the pea MnSOD in transgenic rice plants (T1) was revealed under drought stress induced by polyethylene glycol (PEG) 6000. After PEG treatment the transgenic leaf slices showed reduced electrolyte leakage compared to wild type (WT) leaf slices, whether they were exposed to methyl viologen (MV) or not, suggesting that transgenic plants were more resistant to MV- or PEG-induced oxidative stress. Transgenic plants also exhibited less injury, measured by net photosynthetic rate, when treated with PEG. Our data suggest that SOD is a critical component of the ROS scavenging system in plant chloroplasts and that the expression of MnSOD can improve drought tolerance in rice.     

Invited review: manganese superoxide dismutase in disease

Manganese superoxide dismutase (MnSOD) is essential for life as dramatically illustrated by the neonatal lethality of mice that are deficient in MnSOD. In addition, mice expressing only 50% of the normal compliment of MnSOD demonstrate increased susceptibility to oxidative stress and severe mitochondrial dysfunction resulting from elevation of reactive oxygen species. Thus, it is important to know the status of both MnSOD protein levels and activity in order to assess its role as an important regulator of cell biology.

Numerous studies have shown that MnSOD can be induced to protect against pro-oxidant insults resulting from cytokine treatment, ultraviolet light, irradiation, certain tumors, amyotrophic lateral sclerosis, and ischemia/reperfusion. In addition, overexpression of MnSOD has been shown to protect against pro-apoptotic stimuli as well as ischemic damage. Conversely, several studies have reported declines in MnSOD activity during diseases including cancer, aging, progeria, asthma, and transplant rejection. The precise biochemical/molecular mechanisms involved with this loss in activity are not well understood. Certainly, MnSOD gene expression or other defects could play a role in such inactivation. However, based on recent findings regarding the susceptibility of MnSOD to oxidative inactivation, it is equally likely that post-translational modification of MnSOD may account for the loss of activity. Our laboratory has recently demonstrated that MnSOD is tyrosine nitrated and inactivated during human kidney allograft rejection and human pancreatic ductal adenocarcinoma. We have determined that peroxynitrite (ONOO^-) is the only known biological oxidant competent to inactivate enzymatic activity, to nitrate critical tyrosine residues, and to induce dityrosine formation in MnSOD. Tyrosine nitration and inactivation of MnSOD would lead to increased levels of superoxide and concomitant increases in ONOO^- within the mitochondria which, could lead to tyrosine nitration/oxidation of key mitochondrial proteins and ultimately mitochondrial dysfunction and cell death. This article assesses the important role of MnSOD activity in various pathological states in light of this potentially lethal positive feedback cycle involving oxidative inactivation.     

Role of heme oxygenase-1 in the regulation of manganese superoxide dismutase gene expression in oxidatively-challenged astroglia

Manganese superoxide dismutase (MnSOD) is an antioxidant enzyme that reduces superoxide anion to hydrogen peroxide in cell mitochondria. MnSOD is overexpressed in normal aging brain and in various central nervous system disorders; however, the mechanisms mediating the upregulation of MnSOD under these conditions remain poorly understood. We previously reported that cysteamine (CSH) and other pro-oxidants rapidly induce the heme oxygenase-1 (HO-1) gene in cultured rat astroglia followed by late upregulation of MnSOD in these cells. In the present study, we demonstrate that antecedent upregulation of HO-1 is necessary and sufficient for subsequent induction of the MnSOD gene in neonatal rat astroglia challenged with CSH or dopamine, and in astroglial cultures transiently transfected with full-length human HO-1 cDNA. Treatment with potent antioxidants attenuates MnSOD expression in HO-1-transfected astroglia, strongly suggesting that intracellular oxidative stress signals MnSOD gene induction in these cells. Activation of this HO-1-MnSOD axis may play an important role in the pathogenesis of Alzheimer disease, Parkinson disease and other free radical-related neurodegenerative disorders. In these conditions, compensatory upregulation of MnSOD may protect mitochondria from oxidative damage accruing from heme-derived free iron and carbon monoxide liberated by the activity of HO-1.     

Hydrogen bonding in human manganese superoxide dismutase containing 3-fluorotyrosine

Incorporation of 3-fluorotyrosine and site-specific mutagenesis has been utilized with Fourier transform infrared (FTIR) spectroscopy and x-ray crystallography to elucidate active-site structure and the role of an active-site residue Tyr34 in human manganese superoxide dismutase (MnSOD). Calculated harmonic frequencies at the B3LYP/6-31G** level of theory for L-tyrosine and its 3-fluorine substituted analog are compared to experimental frequencies for vibrational mode assignments. Each of the nine tyrosine residues in each of the four subunits of the homotetramer of human MnSOD was replaced with 3-fluorotyrosine. The crystal structures of the unfluorinated and fluorinated wild-type MnSOD are nearly superimposable with the root mean-square deviation for 198 alpha-carbon atoms at 0.3 A. The FTIR data show distinct vibrational modes arising from 3-fluorotyrosine in MnSOD. Comparison of spectra for wild-type and Y34F MnSOD showed that the phenolic hydroxyl of Tyr34 is hydrogen bonded, acting as a proton donor in the active site. Comparison with crystal structures demonstrates that the hydroxyl of Tyr34 is a hydrogen bond donor to an adjacent water molecule; this confirms the participation of Tyr34 in a network of residues and water molecules that extends from the active site to the adjacent subunit.     

Thermally triggered metal binding by recombinant Thermus thermophilus manganese superoxide dismutase, expressed as the apo-enzyme

Manganese superoxide dismutase from the extremely thermophilic eubacterium Thermus thermophilus has been cloned and expressed at high levels in a mesophilic host (Escherichia coli) as a soluble tetrameric protein mainly present as the metal-free apo-enzyme. Incubation of the purified apo-enzyme with manganese salts at ambient temperature did not restore superoxide dismutase activity, but reactivation could be achieved by heating the protein with Mn(II) at higher temperatures, approaching the physiological growth temperature for T. thermophilus. Heat annealing followed by incubation with manganese at lower temperature fails to reactivate the enzyme, demonstrating that a simple misfolding of the protein is not responsible for the observed behavior. The in vitro metal uptake is nonspecific, and manganese, iron, and vanadium all bind, but only manganese restores catalytic activity. Bound metal ions do not exchange during heat treatment, indicating that the formation of the metal complex is effectively irreversible under these conditions. The metallation process is strongly temperature-dependent, suggesting that substantial activation barriers to metal uptake at ambient temperature are overcome by a thermal transition in the apo-protein structure. A mechanism for SOD metallation is proposed, focusing on interactions at the domain interface.     

Manganese superoxide dismutase from Biomphalaria glabrata

The investigation of the response of Biomphalaria glabrata snails to Echinostoma paraensei (digenea) at 2 days post-exposure by suppression subtractive hybridization yielded a partial sequence of the anti-oxidant enzyme manganese superoxide dismutase (MnSOD). Full-length MnSOD (669nt) from M line and BS-90 strains of B. glabrata differed by one synonymous nucleotide replacement. B. glabrata has 1-4 MnSOD loci (Southern hybridization). Both snail strains expressed MnSOD at equal baseline levels (quantitative PCR). Susceptible snails increased expression of MnSOD following infection with E. paraensei or Schistosoma mansoni, and expression was reduced in the incompatible combination (BS-90 B. glabrata and S. mansoni). Thus, MnSOD did not determine resistance or susceptibility for these parasites, but expression of MnSOD is consistent with its involvement in a stress response of B. glabrata.