Cloning and expression of superoxide dismutase from Mycobacterium bovis BCG

We have previously purified the superoxide dismutase (SOD) of Mycobacterium bovis bacillus Calmette-Guerin (BCG), and there is no signal peptide necessary for protein exportation [S.K. Kang, Y.J. Jung, C.H. Kim, C.Y. Song, Extracellular and cytosolic iron superoxide dismutase from Mycobacterium bovis BCG, Clin. Diagn. Lab. Immunol. 5 (1998) 784-789]. In the present study, SOD gene of M. bovis BCG was cloned and expressed in Escherichia coli, and its complete nucleotide sequence and deduced amino acid composition were determined. The open reading frame from the GTG initiation codon was 621 base pair (bp) in length for the SOD structural gene. The ribosomal-binding sequences (GGAAGG) were 6-12 bp upstream from the initiation codon. The amino acid sequence, deduced from the nucleotide sequence, revealed that the SOD consists of 207 amino acids residues with a molecular weight of 22.8 kDa. The N-terminal amino acid sequence predicted from the nucleotide sequence showed that the structural gene of the SOD is not preceded by leader sequences. There were no cysteine residues in the deduced amino acid composition, indicating that the SOD does not consist of disulfide bonds. Analyses of both nucleotide and amino acid sequences of the SOD showed significant similarity to other pathogenic mycobacterial SODs. Furthermore, the results of fractionation and two-dimensional electrophoresis showed that SOD is also associated with cell membrane, suggesting that there might be a specific mechanism for exportation of SOD in M. bovis BCG as well as other pathogenic mycobacteria. Overexpressed SOD in E. coli was purified from the inclusion bodies, and the histidine tag was removed from the protein using enterokinase. Enzyme activity was then determined by gel staining analysis.     

Combined Proteomic and Molecular Approaches for Cloning and Characterization of Copper–Zinc Superoxide dismutase (Cu, Zn-SOD2) from Garlic (Allium sativum)

Superoxide dismutases (SODs; EC 1.15.1.1) are key enzymes in the cells protection against oxidant agents. Thus, SODs play a major role in the protection of aerobic organisms against oxygen-mediated damages. Three SOD isoforms were previously identified by zymogram staining from Allium sativum bulbs. The purified Cu, Zn-SOD2 shows an antagonist effect to an anticancer drug and alleviate cytotoxicity inside tumor cells lines B16F0 (mouse melanoma cells) and PAE (porcine aortic endothelial cells). To extend the characterization of Allium SODs and their corresponding genes, a proteomic approach was applied involving two-dimensional gel electrophoresis and LC-MS/MS analyses. From peptide sequence data obtained by mass spectrometry and sequences homologies, primers were defined and a cDNA fragment of 456?bp was amplified by RT-PCR. The cDNA nucleotide sequence analysis revealed an open reading frame coding for 152 residues. The deduced amino acid sequence showed high identity (82-87%) with sequences of Cu, Zn-SODs from other plant species. Molecular analysis was achieved by a protein 3D structural model.     

The primary structure of superoxide dismutase purified from anaerobically maintained Bacteroides gingivalis

The superoxide dismutase (SOD) of Bacteroides gingivalis can use either iron or manganese as a cofactor in its catalytic activity. In this study, the complete amino acid sequence of this SOD purified from anaerobically maintained B. gingivalis cells was determined. The proteins consisted of 191 amino acid residues and had a molecular mass of 21,500. The sequence of B. gingivalis SOD showed 44-51% homology with those for iron-specific SODs (Fe-SODs) and 40-45% homology with manganese-specific SODs (Mn-SODs) from several bacteria. However, this sequence homology was considerably less than that seen among the Fe-SOD (65-74%) or Mn-SOD family (42-60%). This indicates that B. gingivalis SOD, which accepts either iron or manganese as metal cofactor, is a structural intermediate between the Fe-SOD and Mn-SOD families.     

Characterization of an iron-containing superoxide dismutase from a higher plant, Citrus limonum

An iron-containing superoxide dismutase (SOD, EC 1.15.1.1) was fully characterized from leaves of the higher plant Citrus limonum R. cv. Verna. This enzyme is the first iron-containing SOD to be characterized in the plant family Rutaceae . The purified Fe-SOD has a molecular mass of about 47 kDa and is composed of two non-covalently joined equal subunits. The amino acid composition determined for the enzyme was compared with that of a wide range of SODs and had highest degree of homology with the Fe-SODs from Brassica campestris and Nuphar luteum . The enzyme was more labile at high temperatures than some eucaryotic and procaryotic Fe-SODs. It showed a maximum stability at pH 7.8. The sensitivity of the enzyme to cyanide, hydrogen peroxide and o -phenanthroline was similar to those reported for other Fe-SODs. but the lemon enzyme was comparatively resistant to H₂O₂. By kinetic competition experiments, the rate constant for the disproportionation of superoxide radicals by lemon Fe-SOD was found to be 1.9 × 10⁹ M ⁻¹ s⁻¹ at pH 7.8 and 25°C. A comparative study between the molecular properties of this higher plant Fe-SOD and SODs from different origins is presented.     

Daily rhythm of MnSOD in the C<Subscript>3</Subscript>-CAM intermediate <Emphasis Type="Italic">Clusia fluminensis</Emphasis> Planch. et Triana.

The C₃-CAM intermediate plant Clusia fluminensis under well-watered at low light conditions opens stomata during the light period. In leaf extracts of this plant we have found two copper-zinc superoxide dismutases (CuZnSODs) and two manganese SODs: MnSOD-like protein (MnSOD II) and MnSOD I. Daily rhythm of the MnSOD I shows maximum activity during the afternoon hours and it is accompanied by only a very small tendency to increase in catalase (CAT) activity and lowering of citrate level.     

Purification of Two Superoxide Dismutase Isozymes and Their Subcellular Localization in Needles and Roots of Norway Spruce (Picea abies L.) Trees

Two isozymes of superoxide dismutase (SOD; EC 1.15.1.1) were purified from Norway spruce (Picea abies L.) needles to apparent electrophoretic homogeneity. Purification factors were 354 for SOD I and 265 for SOD II. The native molecular mass of both purified enzymes was approximately 33 kD, as determined by gel filtration. The subunit molecular weights, as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, were 20,000 for SOD I and 16,000 for SOD II in the presence of 2-mercaptoethanol, and 15,800 and 15,000, respectively, in its absence. These results indicate that the native enzymes were homodimers whose subunits contained intrachain disulfide bonds. Isoelectric points determined by nondenaturing isoelectric focusing were 4.5 and 5.5 for SOD I and II, respectively. NH₂-terminal sequence analysis of the first 22 to 23 amino acids revealed 70 to 75% sequence identity with chloroplastic CuZn SODs from other plant species for SOD I, and 75% sequence identity with the cytosolic CuZn SOD from Scots pine for SOD II. SOD I was the major activity in needles and it was associated with chloroplasts. SOD II activity was dominant in roots.     

耐辐射奇球菌超氧化物歧化酶基因的克隆与序列分析

By using a 453 bp length gene fragment of superoxide dismutase(SOD)as a probe,which was firstly amplified from Deinococcus radiodurans genomic DNA by PCR with degenerate oligonucleotide primers corresponding to the conservative regions of known SODs,a putative SOD gene was identified from the database of D.radiodurans whole genome.Its 636 bp length open reading frame and 5′ and 3′ flanking sequence was determined.The conventional E.coli ribosomal and RNA polymerase binding sites were found upstream from SOD encoding region and an inverted repeat sequence downstream of the termination codon.The deduced 211 amino acid sequence of the structural gene showed a high similarity to other manganese and iron containing SODs in normally conserve regions.     

Copper-zinc superoxide dismutase of Caulobacter crescentus: cloning, sequencing, and mapping of the gene and periplasmic location of the enzyme.

Although widely found in the cytoplasm of eucaryotes, the copper-zinc form of superoxide dismutase (CuZnSOD) has been identified in only a small number of bacterial species. One species is the freshwater bacterium Caulobacter crescentus, which also contains an SOD with iron as the metal cofactor (FeSOD). To investigate the function of this CuZnSOD and its structural relationship to the eucaryotic CuZnSODs, the gene encoding CuZnSOD (sodC) of C. crescentus CB15 was cloned and sequenced. By hybridization to pulsed-field electrophoresis gels, sodC was mapped near cysE in the C. crescentus chromosome. Through analysis of spheroplasts, the two SODs of C. crescentus were shown to be differently localized, CuZnSOD in the periplasm and FeSOD in the cytoplasm. In its natural habitat, C. crescentus is frequently associated with blue-green algae (cyanobacteria). The oxygen evolved by these photosynthetic algae may create an extracellular oxidative stress against which the periplasmic CuZnSOD may defend more effectively than the cytoplasmic FeSOD. Amino acid sequence alignments of C. crescentus CuZnSOD with eucaryotic CuZnSODs and with CuZnSOD of Photobacterium leiognathi (the only other bacterium from which CuZnSOD has been isolated and sequenced) suggest similar supersecondary structures for bacterial and eucaryotic CuZnSODs but reveal four novel substitutions in C. crescentus CuZnSOD: a phenylalanine critical to intrasubunit hydrophobic bonding replaced by alanine, a histidine ligand of zinc replaced by aspartate, and substitutions of two other previously invariant residues that stabilize zinc or both copper and zinc. These amino acid substitutions in C. crescentus CuZnSOD may have implications for its catalysis and stability.     

Characterization of superoxide dismutases purified from either anaerobically maintained or aerated Bacteroides gingivalis.

Superoxide dismutases (SODs) were purified from extracts of either anaerobically maintained or aerated Bacteroides gingivalis. Each purified enzyme (molecular weight, 46,000) was a dimer composed of two subunits of equal sizes. SOD from anaerobically maintained cells (anaero-SOD) contained 1.79 g-atom of Fe and 0.28 g-atom of Mn, and SOD from aerated cells (aero-SOD) contained 1.08 g-atom of Mn and 0.36 g-atom of Fe. Spectral analysis showed that anaero-SOD had the characteristic of Fe-SOD and that aero-SOD had that of Mn-SOD. Both enzyme preparations contained three isozymes with identical isoelectric points. On the basis of inactivation of SOD by H2O2, it was found that aero-SOD consisted of one Mn-SOD and a small quantity of two Fe-SODs, whereas anaero-SOD contained only Fe-SOD. However, each apoprotein from anaero-SOD and aero-SOD, prepared by dialysis in guanidinium chloride plus 8-hydroxyquinoline, showed only one protein band each with the same isoelectric point on an isoelectric focusing gel. Subsequent dialysis of both apoenzymes with either MnCl2 or Fe(NH4)2(SO4)2 restored the activity. These reconstituted SODs showed only one protein band with SOD activity on native polyacrylamide gel electrophoresis. Furthermore, the two enzymes had similar amino acid compositions, and their amino-terminal sequences were identical through the first 12 amino acids. These results suggest that the three isozymes of anaero-SOD and aero-SOD in B. gingivalis are formed from a single apoprotein.     

Superoxide dismutases from the oyster parasite Perkinsus marinus: purification,biochemical characterization,and development of a plate microassay for activity

We have isolated and biochemically characterized superoxide dismutase (SOD) activity in cell extracts of clonally cultured Perkinsus marinus, a facultative intracellular parasite of the Eastern oyster, Crassostrea virginica. In order to assess the SOD activity throughout the purification, we developed and optimized a 96-well-plate microassay based on the inhibition of pyrogallol oxidation. The assay was also adapted to identify SOD activity type (Cu/Zn-, Mn-, or FeSOD), even in mixtures of more than one type of SOD. All SOD activity detected in the cell extracts was of the FeSOD type. Most of the SOD activity in P. marinus trophozoites resides in a major component of subunit molecular weight 24 kDa. The protein was purified by affinity chromatography on an anti-SOD antibody-Sepharose column. Amino-terminal peptide sequence of the affinity-purified protein corresponds to the predicted product of the PmSOD1 gene and indicates that amino-terminal processing has taken place. The results are discussed in the context of processing of mitochondrially targeted SODs.     

Two Fe-superoxide dismutase families respond differently to stress and senescence in legumes

Three main families of SODs in plants may be distinguished according to the metal in the active center: CuZnSODs, MnSOD, and FeSOD. CuZnSODs have two sub-families localized either in plant cell cytosol or in plastids, the MnSOD family is essentially restricted to mitochondria, and the FeSOD enzyme family has been typically localized into the plastid. Here, we describe, based on a phylogenetic tree and experimental data, the existence of two FeSOD sub-families: a plastidial localized sub-family that is universal to plants, and a cytosolic localized FeSOD sub-family observed in determinate-forming nodule legumes. Anti-cytosolic FeSOD (cyt_FeSOD) antibodies were employed, together with a novel antibody raised against plastidial FeSOD (p_FeSOD). Stress conditions, such as nitrate excess or drought, markedly increased cyt_FeSOD contents in soybean tissues. Also, cyt_FeSOD content and activity increased with age in both soybean and cowpea plants, while the cyt_CuZnSOD isozyme was predominant during early stages. p_FeSOD in leaves decreased with most of the stresses applied, but this isozyme markedly increased with abscisic acid in roots. The great differences observed for p_FeSOD and cyt_FeSOD contents in response to stress and aging in plant tissues reveal distinct functionality and confirm the existence of two immunologically differentiated FeSOD sub-families. The in-gel FeSOD activity patterns showed a good correlation to cyt_FeSOD contents but not to those of p_FeSOD. This indicates that cyt_FeSOD is the main active FeSOD in soybean and cowpea tissues. The diversity of functions associated with the complexity of FeSOD isoenzymes depending of the location is discussed.     

Molecular evolution and structure--function relationships of the superoxide dismutase gene families in angiosperms and their relationship to other eukaryotic and prokaryotic superoxide dismutases

This study assesses whether the phylogenetic relationships between SODs from different organisms could assist in elucidating the functional relationships among these enzymes from evolutionarily distinct species. Phylogenetic trees and intron positions were compared to determine the relationships among these enzymes. Alignment of Cu/ZnSOD amino acid sequences indicates high homology among plant sequences, with some features that distinguish chloroplastic from cytosolic Cu/ZnSODs. Among eukaryotes, the plant SODs group together. Alignment of the Mn and FeSOD amino acid sequences indicates a higher degree of homology within the group of MnSODs (>70%) than within FeSODs (approximately 60%). Tree topologies are similar and reflect the taxonomic classification of the corresponding species. Intron number and position in the Cu/Zn Sod genes are highly conserved in plants. Genes encoding cytosolic SODs have seven introns and genes encoding chloroplastic SODs have eight introns, except the chloroplastic maize Sod1, which has seven. In Mn Sod genes the number and position of introns are highly conserved among plant species, but not among nonplant species. The link between the phylogenetic relationships and SOD functions remains unclear. Our findings suggest that the 5' region of these genes played a pivotal role in the evolution of function of these enzymes. Nevertheless, the system of SODs is highly structured and it is critical to understand the physiological differences between the SODs in response to different stresses in order to compare their functions and evolutionary history.     

Complete Amino Acid Sequence of a Copper/Zinc-Superoxide Dismutase from Ginger Rhizome

Superoxide dismutase (SOD) is an antioxidant enzyme protecting cells from oxidative stress. Ginger (Zingiber officinale) is known for its antioxidant properties, however, there are no data on SODs from ginger rhizomes. In this study, we purified SOD from the rhizome of Z. officinale (Zo-SOD) and determined its complete amino acid sequence using N terminal sequencing, amino acid analysis, and de novo sequencing by tandem mass spectrometry. Zo-SOD consists of 151 amino acids with two signature Cu/Zn-SOD motifs and has high similarity to other plant Cu/Zn-SODs. Multiple sequence alignment showed that Cu/Zn-binding residues and cysteines forming a disulfide bond, which are highly conserved in Cu/Zn-SODs, are also present in Zo-SOD. Phylogenetic analysis revealed that plant Cu/Zn-SODs clustered into distinct chloroplastic, cytoplasmic, and intermediate groups. Among them, only chloroplastic enzymes carried amino acid substitutions in the region functionally important for enzymatic activity, suggesting that chloroplastic SODs may have a function distinct from those of SODs localized in other subcellular compartments. The nucleotide sequence of the Zo-SOD coding region was obtained by reverse-translation, and the gene was synthesized, cloned, and expressed. The recombinant Zo-SOD demonstrated pH stability in the range of 5–10, which is similar to other reported Cu/Zn-SODs, and thermal stability in the range of 10–60?°C, which is higher than that for most plant Cu/Zn-SODs but lower compared to the enzyme from a Z. officinale relative Curcuma aromatica.     

Peroxisomal manganese superoxide dismutase: Purification and properties of the isozyme from pea leaves

The peroxisomal manganese superoxide dismutase (perMn‐SOD; EC 1.15.1.1) was purified to homogeneity for the first time from peroxisomes of pea ( Pisum sativum L.) leaves. Peroxisomes were isolated from pea leaves by sucrose density‐gradient centrifugation, and then perMn‐SOD was purified from these organelles by two purification steps involving anion‐exchange and gel‐filtration fast protein liquid chromatography. Pure peroxisomal Mn‐SOD had a specific activity of 2 880 units per mg protein and was purified 3 000‐fold, with a yield of about 7 µg enzyme per kg pea leaves. The relative molecular mass determined for perMn‐SOD was 92 000, and it was composed of four equal subunits of 27 kDa. Ultraviolet and visible absorption spectra of the enzyme showed two absorption maxima at 278 and 483 nm, respectively, and two shoulders at 290 and 542 nm. By isoelectric focusing (pH 5‐7), an isoelectric point of 5.53 was determined for perMn‐SOD. In immunoblot assays, purified Mn‐SOD was recognized by a polyclonal antibody against mitochondrial Mn‐SOD (mitMn‐SOD) from pea leaves. The amino acid sequence of the N‐terminal region of the purified peroxisomal enzyme was determined. A 100% identity was found with the mitMn‐SOD from pea leaves, and high identities were also found with Mn‐SODs from other plant species.     

Unique characteristics of superoxide dismutase of a strictly anaerobic archaebacterium Methanobacterium thermoautotrophicum

The superoxide dismutase (SOD) gene of Methanobacterium thermoautotrophicum (Takao, M., Oikawa, A., and Yasui, A. (1990) Arch. Biochem. Biophys. 283, 210-216), a strictly anaerobic archaebacterium, was expressed in Escherichia coli. The gene product accounted for more than 30% of the host's soluble protein. The purified protein was an active iron-containing tetrameric SOD with specific activity similar to known manganese-containing SODs (MnSODs) of aerobic archaebacteria. Although M. thermoautotrophicum SOD is an iron-containing SOD (FeSOD), it resembles MnSODs in amino acid sequence as judged by criteria distinguishing FeSODs from MnSODs. Moreover, M. thermoautotrophicum SOD is resistant to azide and hydrogen peroxide as MnSODs are, suggesting that its evolution is distinct from known eubacterial FeSODs.     

In silico analyses of superoxide dismutases (SODs) of rice (Oryza sativa L.)

Superoxide dismutases (SODs), members of the metalloenzymes family are most effective intracellular enzymatic antioxidant in aerobic organisms. These enzymes provide the first line of defense in plants against the toxic effects of elevated levels of reactive oxygen species (ROS) generated during various environmental stresses. The availability of high-throughput computational tools has provided better opportunities to characterize the protein features and determine their function. In the present study an attempt was made to gain an insight into the structure and evolution of subunits of SODs (Cu-Zn, Mn and Fe SODs) of rice. The 3-Dimensional structures of SODs were modeled based on available X-ray crystal structures and further validated. The primary sequence, secondary and tertiary structure analysis revealed Mn and Fe SOD to be structurally homologous while Cu-Zn SOD is un-related to either of them. Comparative structural study also revealed former two were dominated by α-helices followed by β-strands in contrast; Cu-Zn SOD dominated by β-strands. Molecular phylogeny indicated a common evolutionary origin of Mn and Fe SOD while Cu-Zn SOD may have evolved separately.