Eukaryotic Superoxide Dismutases Research Articles

Genome-Wide Assessment of Putative Superoxide Dismutases in Unicellular and Filamentous Cyanobacteria

Abstract Cyanobacteria are photoautotrophic prokaryotes capable to grow in diverse ecological habitats, originated 2.5-3.5 billion years ago and were first to produce oxygen. Since then superoxide dismutases (SOD) acquired great significance due to their ability to catalyze detoxification of byproducts of oxygenic photosynthesis i.e. superoxide radicals. In the present study, we extracted information regarding SODs from species of sequenced cyanobacteria and investigated their diversity, conservation, domain structure, and evolution. 144 putative SOD homologs were identified. Unlike other protein families (ex. serine-threonine kinases) SODs are present in all cyanobacterial species reflecting their significant role in survival. However, their distribution varies fewer (0.01%-0.09%) found in unicellular marine strains whereas abundant (0.02%-0.07%) in filamentous nitrogen-fixing cyanobacteria. They were classified into three major subfamilies according to their domain structures: Fe/MnSOD, Cu/ZnSOD and NiSOD. Interestingly, they lack additional domains as found in proteins of other families however motifs and invariant amino acids typical in eukaryotic SODs were conserved well in these proteins indicating similar catalytic mechanism as eukaryotic SODs. Phylogenetic relationships correspond well with phylogenies based on 16S rRNA and clustering occurs on the basis of structural characteristics such as domain organization. Gene gain-and-loss is insignificant during SOD evolution as evidenced by the absence of additional domain. This study has not only examined an overall background of sequence-structure-function interactions for the SOD gene family but also revealed variation among SOD distribution based on ecophysiological and morphological characters.

Unique structural features of the monomeric Cu,Zn superoxide dismutase from Escherichia coli, revealed by X-ray crystallography

The first three-dimensional structure of a functional monomeric Cu,Zn superoxide dismutase (from Escherichia coli, E_SOD) is reported at 2.0 Å resolution ( R-factor=16.8%). Compared to the homologous eukaryotic enzymes, E_SOD displays a perturbed antiparallel β-barrel structure. The most striking structural features observed include extended amino acid insertions in the surface 1,2-loop and S-S subloop, modification of the disulfide bridge connection, and loss of functional electrostatic residues, suggesting a modified control of substrate steering toward the catalytic center. The active site Cu 2+displays a distorted coordination sphere due to an unusually long bond to the metal-bridging residue His61. Inspection of the crystal packing does not show regions of extended contact indicative of a dimeric assembly. The molecular surface region involved in subunit dimerization in eukaryotic superoxide dismutases is structurally altered in E_SOD and displays a net polar nature.

Purification of pro- and eukaryotic superoxide dismutases by charge-controlled hydrophobic chromatography

The process of purifying superoxide dismutases was simplified using charge-controlled hydrophobic chromatography on 10-carboxydecyl Sepharose. In only one chromatographic step following ammonium sulphate precipitation, Fe-containing superoxide dismutase from Pseudomonas putida and Cu,Zn-containing superoxide dismutase from bovine erythrocytes were purified with an overall yield of about 70% to electrophoretic homogeneity. The specific activities of the crystalline enzyme preparations were expressed in McCord and Fridovich units and were 3000 and 3200 U/mg, respectively.

Chemical aspects of structure, function and evolution of superoxide dismutases

The superoxide dismutases (SOD) constitute a class metalloproteins having either Cu Zn , Mn or Fe as their prosthetic group and their function is to dismute O 2 to H 2O 2 and O 2. The distribution of the SODs has to be considered in the light of the acquisition of a permanent defence by organisms against any form of toxicity arising from the increase, by photosynthetic organisms, at the atmospheric oxygen. Anaerobic sulphate reducing bacteria and fermentative anaerobic bacteria were found to contain the enzyme, probably the iron form. This is considered to be the most primitive form of SOD. In general terms the distribution of SOD can be stated to be that the Cu Zn Zn enzyme is essentially eukaryotic enzyme and the iron enzyme is essentially a prokaryotic enzyme. The manganese enzyme can also be considered to be a prokaryotic enzyme. It is, however, also found in mitochondria. Whilst certain species of bacteria have been found to have one form of SOD i.e., either the Fe or the Mn form, there are species with both forms as well. It appears that which form of the enzyme is present depends on the growth conditions. The phototrophic purple sulfur bacterium Thiocupsa roseopersicina grown in the presence of acetate or glucose contains Fe-SOD. Growth on media with glucose but not acetate leads to the formation of Mn-SOD [1]. The synthesis of either Fe- or Mn-SOD with apparently identical protein moiety by Photobacterium shermanii was found to be conditional on the metal supply [2]. All eukaryotic species have the Cu Zn Zn and Mn form of the enzyme. Some plant species have the Fe form as well whilst two bacterial species have the Cu Zn Zn enzyme [3]. Cu Zn Zn SOD is a homodimer of about 32,000 daltons. The molecular weight determined for the enzyme from a variety of sources are all substantially in agreement. By contrast, the Mn- and Fe-forms have a slightly higher molecular weight of about 40,000 daltons. Whilst all the Cu Zn Zn and the Fe-SODs have been shown to be dimeric the manganese enzyme has been found to form tetrameters. The extent of polymerisation does not apparently depend of the source of the enzyme. The Cu Zn Zn SOD contain up to 2 g atoms of both Cu and Zn calculated on the basis of a molecular weight of 32,000 daltons. The values reported in the literature is 1.8 g atoms. The metal content of the Fe and Mn enzymes varies between 1 and 2 g atoms per dimer. Whether this variation is due to loss of metal content can only be determined once the number of metal binding sites is determined from the X-ray structure. Only preliminary data has so far been presented on the X-ray structure for this protein. The complete sequence has only been determined for the Mn enzyme from E. coli and B. stearothermophilus and yeast mitochondria [4-6]. The sequence homology is low compared to the homologies observed between Cu Zn Zn enzyme. The nature of the Mn and Fe ligands is as yet unknown. Preliminary spectroscopy evidence, however, indicates that a tyr could not be a ligand. The Cu Zn Zn SODs have been more extensively investigated than the Fe- and Mn-SODs. The primary structure has been determined for the enzyme from bovine [7] and human erythrocytes [8, 9], horse liver [10], yeast [11, 12], swordfish liver [13] and the free living symbiotic bacterium Photobacterium leiognathi [14]. Extensive homology has been found to occur between the eukaryotic enzymes. The presence of a Cu Zn Zn SOD in P. leiognathi has led to speculation about a possible gene transfer from a eukaryotic to a prokaryotic species. However, the amino acid sequence only shows a 20-25% structural homology between this enzyme and the other eukaryotic SODs indicating an independent evolutionary line. All the Cu Zn Zn SODs have, however, a conserved metal binding site determined from the X-ray structure of the bovine enzyme. These are the histidines 47, 49, 76, 81, 134 for the Cu site and histidines 76, 85, 94 and aspartate 97 for the zinc site. Arginine 157 claimed to be essential for activity is also conserved. However cysteine 125 is only present in the human enzyme.