Structural identity between the iron- and manganese-containing superoxide dismutases

We have recently reported the first complete amino acid sequence of an iron-containing superoxide dismutase. The iron enzyme is thought to be closely homologous to the manganese-containing superoxide dismutases. The availability of complete amino acid sequence information for four manganese superoxide dismutases and the crystal structures for two iron and two manganese superoxide dismutases prompted us to investigate the degree of homology between the two proteins at various levels. We report that it is not possible to clearly distinguish the two proteins on the basis of their secondary or tertiary structures. It would appear that a small number of single site substitutions are responsible for conferring distinguishing properties between the two proteins. Substitution of glycine 77 and glutamine 154 by a glutamine and an alanine respectively in Photobacterium leiognathi iron superoxide dismutase may distinguish the kinetic and other particular properties of this protein from the manganese protein (and other iron superoxide dismutases). Furthermore the primary structure of both the iron and manganese proteins does not appear to have any homology with any other known amino acid sequence.     

Primary structure of copper-zinc superoxide dismutase from Neurospora crassa

The complete amino acid sequence of copper-zinc superoxide dismutase from Neurospora crassa is reported. The subunit consists of 153 amino acids and has a Mr of 15,850. The primary structure was determined by automated and manual sequence analysis of peptides obtained by digestions of the carboxymethylated and aminoethylated enzyme with trypsin and thermolysin. The protein is devoid of tryptophan and methionine and displays a free amino terminus. Comparison of the amino acid sequence with those from human erythrocyte, bovine erythrocyte, horse liver, swordfish liver, and yeast copper-zinc superoxide dismutases reveals a high degree of sequence homology among the six enzymes. Most prominently, the regions containing the amino acid residues participating in the metal-binding and the half-cystine residues forming the intramolecular disulfide bridge are highly conserved. The invariant amino acids Pro 74 and Asp 76 of the four vertebrate and yeast superoxide dismutases were found to be substituted by arginine and alanine, respectively, in the Neurospora enzyme. These radical substitutions occurring in the zinc ligand region, known to form a characteristic loop structure in bovine erythrocyte copper-zinc superoxide dismutase (Tainer, J. A., Getzoff, E. D., Beem, K. M., Richardson, J. S., and Richardson, D. C. (1982) J. Mol. Biol. 160, 181-217), however, do not affect the catalytic properties of the Neurospora enzyme.     

A tetrameric iron superoxide dismutase from the eucaryote Tetrahymena pyriformis

An iron-containing superoxide dismutase has been purified from the protozoan Tetrahymena pyriformis. It has a molecular weight of 85,000 and is composed of four subunits of equal size. The tetramer contains 2.5 g atoms of ferric iron. Visible absorption and electron spin resonance spectra closely resemble those of other iron-containing superoxide dismutases. The amino acid sequence of the iron superoxide dismutase was determined. Each subunit is made up of 196 residues, corresponding to a molecular weight of 22,711. Comparison of the primary structure with the known sequences of other iron-containing superoxide dismutases reveals a relatively low degree of identity (33-34%). However, a higher percentage identity is found with mammalian manganese-containing superoxide dismutases (41-42%). The amino acid sequence is discussed in consideration of residues that may distinguish iron from manganese or dimeric from tetrameric superoxide dismutases.     

The amino-acid sequence of copper/zinc superoxide dismutase from swordfish liver. Comparison of copper/zinc superoxide dismutase sequences

The amino acid sequence of copper/zinc superoxide dismutase from swordfish (Xiphias gladius) liver has been determined by alignment of the tryptic peptides according to the known sequence of bovine erythrocyte copper/zinc superoxide dismutase. This alignment has resulted in the ligands to the copper (His-47, 49, 76 and 94) and the zinc (His-76, 85, 134 and Asp-97) being conserved in all the copper/zinc superoxide dismutases sequenced so far. Also conserved in the sequences are the cysteines forming the intrachain disulphide bridge (Cys-58 and 160) and the essential arginine (Arg-157). Comparison of the amino acid sequence of swordfish liver copper/zinc superoxide dismutase with the bovine, human, horse, yeast and Photobacterium leiognathi indicates that the swordfish enzyme has a high homology with the other eukaryotic enzymes. Low homology is, however, observed with the P. leiognathi enzyme.     

The amino acid sequence of mangano superoxide dismutase from Escherichia coli B.

The complete amino acid sequence of the mangano superoxide dismutase from Escherichia coli B has been deduced through characterization of peptides from cyanogen bromide, bromonitrophenylsulfenyl skatole, citraconylated tryptic, and succinylated tryptic digests of the intact polypeptide chain and through subfragmentation of selected peptides with chymotrypsin, thermolysin, trypsin, and Staphylococcus aureus V8 extracellular protease. No significant homology is detected on comparison with the sequence of the copper- and zinc-containing superoxide dismutase from bovine erythrocytes, indicating that the manganese-iron and the copper-zinc classes of dismutases arose from independent evolutionary ancestors, a proposal previously based solely on enzymological and NH2-terminal sequence data. The amino acid sequence listed below corresponds to a molecular weight of 22,900 and appears to be identical in each subunit polypeptide of the native enzyme dimer. formula: (see text).     

Amino acid sequence of iron-superoxide dismutase from Pseudomonas ovalis

The amino acid sequence of iron-superoxide dismutase from Pseudomonas ovalis was deduced by the analyses of peptides derived from limited hydrolysis of the aminoethylated or pyridylethylated apoprotein with trypsin, Staphylococcus aureus V8 protease, and dilute acid hydrolysis. The polypeptide chain contains 195 amino acid residues and has a calculated Mr of 21,421. The sequence is highly homologous (65% identity) to the recently published sequence of the iron-superoxide dismutase from Photobacterium leiognathi. It is also homologous to the known sequences of the manganese-superoxide dismutase by sharing 33-53% identical residues. Alignment of the superoxide dismutase sequences and the available structural information from X-ray crystallography suggest that the ligands to the iron in the P. ovalis superoxide dismutase are His-26, His-74, Asp-156 and His-160, which align with the ligands to the manganese in the Thermus thermophilus manganese-superoxide dismutase. The sequence information of the P. ovalis dismutase will facilitate refinement of the X-ray crystallographic data that are now available at 2.9 A resolution.     

The primary structure of human liver manganese superoxide dismutase

The complete amino acid sequence of manganese superoxide dismutase from human liver was determined. The sequence was deduced following characterization of the peptides obtained from tryptic, chymotryptic, and Staphylococcus aureus digests of the apoprotein. Chemical cleavage with dimethyl sulfoxide-hydrobromic acid was also carried out. The amino acid sequence listed below is made up of 196 amino acids and the two subunit polypeptides in the native enzyme appear to be identical. No homology was observed with copper/zinc containing class of superoxide dismutase. Lys-His-Ser-Leu-Pro-Asp-Leu-Pro-Tyr-Asp-Tyr-Gly-Ala-Leu-Glu-Pro-His-Il e -Asn-Ala-Gln-Ile-Met-Gln-Leu-His-His-Ser-Lys-His-His-Ala-Ala-Tyr-Val-Asn -Asn-Leu-Asn-Val-Thr-Gln-Glu-Lys-Tyr-Gln-Glu-Ala-Leu-Ala-Lys-Gly-Asp-Val -Thr-Ala-Gln-Ile-Ala-Leu-Gln-Pro-Ala-Leu-Lys-Phe-Asn-Gly-Gly-Gly-His-Ile -Asn-His-Ser-Ile-Phe-Trp-Thr-Asn-Leu-Ser-Pro-Asn-Gly-Gly-Gly-Gln-Pro-Lys -Gly-Glu-Leu-Leu-Glu-Ala-Ile-Lys-Arg-Asp-Phe-Gly-Ser-Phe-Asp-Lys-Phe-Lys -Gln-Lys-Leu-Thr-Ala-Ala-Ser-Val-Gly-Val-Gln-Gly-Ser-Gly-Trp-Leu-Gly-Phe -Asn-Lys-Gln-Arg-Gly-His-Leu-Gln-Ile-Ala-Ala-Cys-Pro-Asn-Gln-Asp-Pro-Leu -Gln-Gly-Thr-Thr-Gly-Leu-Ile-Pro-Leu-Leu-Gly-Ile-Asp-Val-Trp-Glu-His-Ala -Tyr-Tyr-Leu-Gln-Tyr-Lys-Asn-Val-Arg-Pro-Asp-Tyr-Leu-Lys-Ala-Ile-Trp-Asn -Val-Ile-Asn-Trp-Glu-Asn-Val-Thr-Glu-Arg-Tyr-Met-Ala-Cys-Lys-Lys.     

Characterization of cDNAs encoding CuZn-superoxide dismutases in Scots pine

A Scots pine (Pinus sylvestris L.) cDNA library was screened with two heterologous cDNA probes (P31 and T10) encoding cytosolic and chloroplastic superoxide dismutases (SOD) from tomato. Several positive clones for cytosolic and chloroplastic superoxide dismutases were isolated, subcloned, mapped and sequenced. One of the cDNA clones (PS3) had a full-length open reading frame of 465 bp corresponding to 154 amino acid residues and showed approximately 85% homology with the amino acid sequences of angiosperm cytosolic SOD counterparts. Another cDNA clone (PST13) was incomplete, but encoded a putative protein with 93% homology to pea and tomato chloroplastic superoxide dismutase. The derived amino acid sequence from both cDNA clones matched the corresponding N-terminal amino acid sequence of the purified mature SOD isozymes. Northern blot hybridizations showed that, cytosolic and chloroplastic CuZn-SOD are expressed at different levels in Scots pine organs. Sequence data and Southern blot hybridization confirm that CuZn-SODs in Scots pine belong to a multigene family. The results are discussed in relation to earlier observations of CuZn-SODs in plants.     

The amino-acid sequence of rabbit Cu-Zn superoxide dismutase

The primary structure of Cu-Zn superoxide dismutase from rabbit liver was investigated. The reduced and S-carboxymethylated enzyme was treated with cyanogen bromide, trypsin or Staphylococcus aureus proteinase V8. The resulting peptides were separated by high-performance liquid chromatography and sequenced by automated Edman degradation. With the exception of the N- and C-terminus the complete sequence was established by means of overlapping peptides. The N-terminus is blocked and thus not susceptible to Edman degradation. The amino-acid composition of the tryptic N-terminal peptide corresponds to that of the cytoplasmatic Cu-Zn superoxide dismutases of other mammals investigated. The chromatographic behaviour of these N-terminal peptides on a reversed phase C18 column is also identical, thus suggesting also for the rabbit Cu-Zn superoxide dismutase the N-terminal sequence Ac-Ala-Thr-Lys. The C-terminus was demonstrated to have the sequence -Ile-Ala-Pro by enzymatic degradation with carboxypeptidase Y. The complete amino-acid sequence of the rabbit Cu-Zn superoxide dismutase consists of 152 amino-acids and shows the expected homology to other Cu-Zn enzymes published so far. The aspartate and six histidine residues known to complex the metal ions are conserved at homologous positions. This also applies for the arginine residue near the C-terminus which is supposed to direct the anionic superoxide radical towards the active centre of the enzyme. The amino acid sequence of the rabbit Cu-Zn superoxide dismutase corresponds to those of other mammals in more than 80% of its amino-acid residues. From a total of 152 amino-acid residues the rabbit shares with rat 128, with mouse 130, with horse 127, with pig 126/127, with cattle 130 and with man 131 amino acids in homologous positions. However the Cu-Zn superoxide dismutases of closely related mammals like rats and mice differ in only five amino acid residues of their sequence. A phylogenetic closer relatedness between lagomorphs and rodents than between other orders of mammals, could not be derived from the sequence data given. Rather rodents and lagomorphs are to be considered as two evolutionary independent orders of mammals.     

Primary structure of Cu-Zn superoxide dismutase of Brassica oleracea proves homology with corresponding enzymes of animals, fungi and prokaryotes

The complete amino-acid sequence of Cu-Zn superoxide dismutase from white cabbage (Brassica oleracea) is reported. The polypeptide chain consists of 151 amino acids and has a molecular mass of 15,604 Da. The primary structure of the reduced and S-carboxymethylated protein was determined by automated solid phase sequence analysis of tryptic fragments and peptides obtained by digestion with Staphylococcus aureus proteinase V8. The protein shows a free amino terminus as was found for all non-mammalian Cu-Zn enzymes so far sequenced. Comparison of the amino-acid sequence from the plant Cu-Zn enzyme with those from nine eukaryotic enzymes reveals a high degree of homology (50-64%) among these enzymes. As already described for all the eukaryotic Cu-Zn superoxide dismutases also the plant enzyme shows a low homology (about 28%) with the bacteriocuprein of Photobacterium leiognathi. However, the amino-acid residues involved in metal binding, the half-cystine residues forming the intermolecular disulfide bridge, one of the arginine and some glycine and proline residues are conserved in all eleven Cu-Zn superoxide dismutases. Although the precise role of the 23 completely conserved residues is not yet completely understood, they appear to almost define the minimum structural requirements for optimizing the superoxide dismutation at the catalytic site, since functional differences between the eleven enzymes are not detectable.     

Characterization of a Manganese Superoxide Dismutase from the Higher Plant Pisum sativum

A manganese-containing superoxide dismutase (EC 1.15.1.1) was fully characterized from leaves of the higher plant Pisum sativum L., var. Lincoln. The amino acid composition determined for the enzyme was compared with that of a wide spectrum of superoxide dismutases and found to have a highest degree of homology with the mitochondrial manganese superoxide dismutases from rat liver and yeast. The enzyme showed an apparent pH optimum of 8.6 and at 25°C had a maximum stability at alkaline pH values. By kinetic competition experiments, the rate constant for the disproportionation of superoxide radicals by pea leaf manganese superoxide dismutase was found to be 1.61 × 10⁹ molar⁻¹·second⁻¹ at pH 7.8 and 25°C. The enzyme was not sensitive to NaCN or to H₂O₂, but was inhibited by N₃⁻. The sulfhydryl reagent p-hydroxymercuribenzoate at 1 mm concentration produced a nearly complete inhibition of the manganese superoxide dismutase activity. The metal chelators o-phenanthroline, EDTA, and diethyldithiocarbamate all inhibited activity slightly in decreasing order of intensity. A comparative study between this higher plant manganese superoxide dismutase and other dismutases from different origins is presented.     

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.     

Multiple forms of extracellular superoxide dismutase in needles, stem tissues and seedlings of Scots pine

Extracellular superoxide dismutase was found in extra-xylematic bark tissues and seedlings of Pinus sylvestris L. The isozymes were partially purified by infiltration and analysed by polyacrylamide gel electrophoresis. One of the major isoforms was purified to homogeneity and the N-terminal amino acid sequence was determined. The isoelectric point of these extra-cellular forms is distinctly higher than that of cellular superoxide dismutases. Furthermore, a novel type of superoxide dismutase with an isoelectric point higher than nine was detected in a crude needle extract.Keywords: Superoxide dismutase, apoplasm, seedlings, oxidative burst, Pinus sylvestris L.      

Iron-containing superoxide dismutase from Crithidia fasciculata. Purification, characterization, and similarity to Leishmanial and trypanosomal enzymes

Leishmania tropica, Trypanosoma brucei, Trypanosoma cruzi, and Crithidia fasciculata have superoxide dismutases which are insensitive to cyanide and sensitive to peroxide and azide, properties characteristic of iron-containing superoxide dismutase. Studies on the superoxide dismutase of C. fasciculata have revealed that: 1) the enzyme is located in the cytosol; 2) isozymes exist; 3) the major superoxide dismutase isozyme (superoxide dismutase 2) has Mr approximately equal to 43,000 and consists of two equal-sized subunits, each of which contains 1.4 atoms of iron. Comparisons of the amino acid content of this crithidial superoxide dismutase with those of superoxide dismutases from other sources suggests that the crithidial enzyme is closely related to bacterial Fe-containing superoxide dismutases, and only distantly related to human Mn- and Cu,Zn-containing superoxide dismutases and to Euglena Fe-containing superoxide dismutase. Attempts are now underway to develop specific inhibitors of the trypanosomatid superoxide dismutase which may be of use in the treatment of leishmaniasis or trypanosomiasis.     

Primary structure and properties of Mn-superoxide dismutase from scallop adductor muscle

Manganese-superoxide dismutase was purified to homogeneity from scallop adductor muscle using DEAE-Sephacel, Buthyl-Cellulofine and Superdex 200 pg column chromatographies. The molecular weights of the purified enzyme were calculated to be 22,321.4 according to time-of-flight mass spectrometry, and to be approximately 95,000 and 93,000 on Superdex 200 pg column chromatography and non-denatured PAGE, respectively, and were calculated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 24,000 and 25,000 in the absence and 25,000 in the presence of beta-mercaptoethanol. These findings suggested that the native enzyme is composed of four identical subunits. Other properties of scallop adductor muscle manganese-superoxide dismutase, including pH stability and heat stability, were also determined. We determined the partial amino acid sequences of purified manganese-superoxide dismutase using digestions by bromocyan and lysyl endopeptidase and also determined the manganese-superoxide dismutase cDNA structure. The amino acid sequence of the enzyme obtained using both methods showed homology to those of vertebrates such as human, bovine, chicken, Xenopus and zebrafish manganese-superoxide dismutases (64.91, 65.35, 64.47, 63.27 and 64.60%, respectively). We also predicted the 3D structure of scallop adductor muscle manganese-superoxide dismutase using molecular operating environment and compared its structure with those of other manganese-superoxide dismutases. The overall structure of scallop adductor muscle manganese-superoxide dismutase was very similar to those of other species, including human and Aspergillus.     

Purification of a manganese-containing superoxide dismutase from Halobacterium halobium

An oxygen-induced superoxide dismutase was purified from the halophilic bacterium, Halobacterium halobium, strain NRL. Due to the high salt requirement for enzyme stability, the purification had to be performed in the presence of 2 M NaCl. The pI of the protein was 4.95. The approximate Mr was 38,500. The subunit size as determined by sodium dodecyl sulfate-electrophoresis was approximately 19,000. Metal analysis showed 1.5 atoms of manganese per dimer, 0.5 atom zinc, and 1.54 atoms copper. The N-terminal sequence of amino acids was determined, and based upon the first 26 amino acids significant homology to other manganese- and iron-containing superoxide dismutases was revealed.     

Iron superoxide dismutase. Nucleotide sequence of the gene from Escherichia coli K12 and correlations with crystal structures

The nucleotide sequence of the iron superoxide dismutase gene from Escherichia coli K12 has been determined. Analysis of the DNA sequence and mapping of the mRNA start reveal a unique promoter and a putative rho-independent terminator, and suggest that the Fe dismutase gene constitutes a monocistronic operon. The gene encodes a polypeptide product consisting of 192 amino acid residues with a calculated Mr of 21,111. The published N-terminal amino acid sequence of E. coli B Fe dismutase (Steinman, H. M., and Hill, R. L. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 3725-3729), along with the sequences of seven other peptides reported here, was located in the primary structure deduced from the K12 E. coli gene sequence. A new molecular model for iron dismutase from E. coli, based on the DNA sequence and x-ray data for the E. coli B enzyme at 3.1 A resolution, allows detailed comparison of the structure of the iron enzyme with manganese superoxide dismutase from Thermus thermophilus HB8. The structural similarities are more extensive than indicated by earlier studies and are particularly striking in the vicinity of the metal-ligand cluster, which is surrounded by conserved aromatic residues. The combined structural and sequence information now available for a series of Mn and Fe superoxide dismutases identifies variable regions in these otherwise very similar molecules; the principal variable site occurs in a surface region between the two long helices which dominate the N-terminal domain.     

Superoxide Dismutase and Oxygen Metabolism in Streptococcus faecalis and Comparisons with Other Organisms

Streptococcus faecalis contains a single superoxide dismutase that has been purified to homogeneity with a 55% yield. This enzyme has a molecular weight of 45,000 and is composed of two subunits of equal size. It contains 1.3 atoms of manganese per molecule. Its amino acid composition was determined and is compared with that for the superoxide dismutases from Escherichia coli, Streptococcus mutans, and Mycobacterium lepraemurium. When used as an antigen in rabbits, the S. faecalis enzyme elicited the formation of a precipitating and inhibiting antibody. This antibody cross-reacted with the superoxide dismutase present in another strain of S. faecalis, but neither inhibited nor precipitated the superoxide dismutases in a wide range of other bacteria, including several other streptococci, such as S. pyogenes, S. pneumoniae, and S. lactis. The inhibiting antibody was used to suppress the superoxide dismutase activity present in cell extracts of S. faecalis and thus allow the demonstration that 17% of the total oxygen consumption by such extracts, in the presence of reduced nicotinamide adenine dinucleotide, was associated with the production of O(2) (-). A variety of bacterial species were surveyed for their content of superoxide dismutases. The iron-containing enzyme was distinguished from the manganese-containing enzyme through the use of H(2)O(2), which inactivates the former more readily than the latter. Some of the bacteria appeared to contain only the iron enzyme, others only the manganese enzyme, and still others both. Indeed, some had multiple, electrophoretically distinct superoxide dismutases in both categories. There was no discernible absolute relationship between the types of superoxide dismutases in a particular organism and their Gram-stain reaction.     

The amino acid sequences of the copper/zinc superoxide dismutases from swordfish and Photobacter leiognathi confirm the predictions made from the compositions

Recent suggestions that the amino acid sequence of the copper/zinc superoxide dismutases of swordfish and Photobacter leiognathi do not support the theory that the bacterium obtained the gene for the enzyme by transfer from its eucaryotic symbiont [Rocha, H. A., Bannister, W. H. and Bannister, J. V. (1984) Eur. J. Biochem. 145, 477-484] are examined. The amount of difference between the sequence is in good agreement with expectation from the amino acid compositions. Moreover, the gene-transfer hypothesis cannot be discarded without postulating an enormous increase in the rate at which the superoxide dismutase gene has accumulated amino acid substitutions since the divergence of the swordfish and cattle lineages.