Crystallization and preliminary crystallographic analysis of manganese superoxide dismutase from Bacillus halodenitrificans

Manganese superoxide dismutase (GP-MnSOD), a component of the so-called 'green protein' (green protein complex) from the facultative anaerobic halodenitrifier Bacillus halodenitrificans, has been crystallized using the hanging-drop vapor diffusion method. Crystals have unit-cell parameters a=b=93.4 A, c=65.0 A, and belong to the space group P4(3)2(1)2. Preliminary analysis indicates there is one monomer in each asymmetric unit. The structural information from this enzyme will enrich our knowledge on its high catalytic activity and its possible role in green protein complex.     

Characterization of the superoxide dismutase of the denitrifying bacterium,Bacillus halodenitrificans

Summary Bacillus halodenitrificans produced a dimeric, manganese-containing superoxide dismutase constitutively when grown either aerobically or as a denitrifier. The molecular mass of the enzyme was determined by sedimentation equilibrium to be 41.4±3 kDa with each subunit estimated at 26 kDa. Plasma emission spectroscopy indicated the presence of 1.22 mol manganese atoms/mol holoenzyme. The electronic absorption spectrum displayed a broad band centered at approximately 474 nm (=560 mM^–1 · cm^–1) and a shoulder at 595 nm. In the ultraviolet range, the spectrum exhibited split maxima at 278 nm and 283 nm and a shoulder at 291 nm, thus resembling the spectra of superoxide dismutase fromBacillus subtilis andEscherichia coli. The amino acid composition of theB. halodenitrificans enzyme differed slightly quantitatively but little qualitatively from counterpart enzymes from other sources. Like the superoxide dismutases ofMycobacterium lepraemurium and human mitochondria, theB. halodenitrificans enzyme exhibited several cysteine residues. As expected from the capacity superoxide dismutase exhibits for protecting NO as neutrophil cytotoxicity factor, theB. halodenitrificans superoxide dismutase did not interfere with accumulation of NO produced by the organism's nitrite reductase.     

Crystal structure of a nucleoside diphosphate kinase from Bacillus halodenitrificans: coexpression of its activity with a Mn-superoxide dismutase

We found that when grown under anaerobic conditions the moderate halophile, gram-positive bacterium Bacillus halodenitrificans (ATCC 49067) synthesizes large amounts of a polypeptide complex that contains a heme center capable of reversibly bind nitric oxide. This complex, when exposed to air, dissociates and reassociates into two active components, a Mn-containing superoxide dismutase (SOD) and a nucleoside diphosphate kinase (BhNDK). The crystal structure of this latter enzyme has been determined at 2.2A resolution using molecular replacement method, based on the crystal structure of Drosophila melanogaster NDK. The model contains 149 residues of a total 150 residues and 34 water molecules. BhNDK consists of a four-stranded antiparallel beta-sheet, whose surfaces are partially covered by six alpha-helices, and its overall and active site structures are similar to those of homologous enzymes. However, the hexameric packing of BhNDK shows that this enzyme is different from both eukaryotic and gram-negative bacteria. The need for the bacterium to presynthesize both SOD and NDK precursors which are activated during the anaerobic-aerobic transition is discussed.     

Isolation of a germin-like protein with manganese superoxide dismutase activity from cells of a moss, Barbula unguiculata

A novel extracellular Mn-superoxide dismutase (SOD) was isolated from a moss, Barbula unguiculata. The SOD was a glycoprotein; the apparent molecular mass of its native form was 120 kDa, as estimated by gel filtration chromatography, and that of its monomer was 22,072 Da, as estimated by time of flight mass spectroscopy. The protein had manganese with a stoichiometry of 0.80 Mn/monomer. The cDNA clone for a gene encoding the extracellular Mn-SOD was isolated. Sequence analysis showed that it has a strong similarity to germin (oxalate oxidase) and germin-like proteins (GLPs) of several plant species and possesses all the characteristic features of members of the germin family. The clone encoding this extracellular Mn-SOD was therefore designated B. unguiculata GLP (BuGLP). BuGLP had no oxalate oxidase activity. In addition, the cDNA for a gene encoding the moss mitochondrial Mn-SOD was isolated. Its amino acid sequence had little similarity to that of BuGLP, even though a close similarity was observed among the mitochondrial Mn-SODs of various organisms. BuGLP was the first germin-like protein that was really demonstrated to be a metalloprotein with Mn-SOD activity but no oxalate oxidase activity.     

Crystal structure and biochemical characterization of a manganese superoxide dismutase from Chaetomium thermophilum

A manganese superoxide dismutase from the thermophilic fungus Chaetomium thermophilum (CtMnSOD) was expressed in Pichia pastoris and purified to homogeneity. Its optimal temperature was 60 °C with approximately 75% of its activity retained after incubation at 70 °C for 60 min. Recombinant yeast cells carrying C. thermophilum mnsod gene exhibited higher stress resistance to salt and oxidative stress-inducing agents than control yeast cells. In an effort to provide structural insights, CtMnSOD was crystallized and its structure was determined at 2.0 Å resolution. The overall architecture of CtMnSOD was found similar to other MnSODs with highest structural similarities obtained against a MnSOD from the thermotolerant fungus Aspergillus fumigatus. In order to explain its thermostability, structural and sequence analysis of CtMnSOD with other MnSODs was carried out. An increased number of charged residues and an increase in the number of intersubunit salt bridges and the Thr:Ser ratio were identified as potential reasons for the thermostability of CtMnSOD.     

The structure of rabbit extracellular superoxide dismutase differs from the human protein

The cDNA sequence encoding rabbit, mouse, and rat extracellular superoxide dismutase (EC-SOD) predicts that the protein contains five cysteine residues. Human EC-SOD contains an additional cysteine residue and folds into two forms with distinct disulfide bridge patterns. One form is enzymatically active (aEC-SOD), while the other is inactive (iEC-SOD). Due to the lack of the additional cysteine residue rabbit, mouse, and rat EC-SOD are unable to generate an inactive fold identical to human iEC-SOD. The amino acid sequences predict the formation of aEC-SOD only, but other folding variants cannot be ruled out based on the heterogeneity observed for human EC-SOD. To test this, we purified EC-SOD from rabbit plasma and determined the disulfide bridge pattern. The results revealed that the disulfide bridges are homogeneous and identical to human aEC-SOD. Four cysteine residues are involved in two intra-disulfide bonds while the C-terminal cysteine residue forms an intersubunit disulfide bond. No evidence for other folding variants was detected. These findings show that rabbit EC-SOD exists as an enzymatically active form only. The absence of iEC-SOD in rabbits suggests that the structure and aspects of the physiological function of EC-SOD differs significantly between rabbit and humans. This is an important notion to take when using these animals as model systems for oxidative stress.     

Amino-acid sequence of a tetrameric, manganese superoxide dismutase from Thermus thermophilus HB8

The amino-acid sequence of a tetrameric manganese superoxide dismutase from Thermus thermophilus HB8 has been determined. The protein was cleaved with cyanogen bromide (BrCN) into four peptides and their alignment was deduced through the fragment of partial cleavage with BrCN and the peptides were produced by cleavage of the protein with o-iodosobenzoic acid. Most of the peptides were sequenced by solid phase Edman degradation. Some of the peptides were sequenced by the Edman dansyl method after sub-fragmentation by proteinase digestion. The amino-acid sequence consists of 203 residues corresponding to a subunit molecular weight of 23,144.     

Novel mechanisms for superoxide-scavenging activity of human manganese superoxide dismutase determined by the K68 key acetylation site

Superoxide is the primary reactive oxygen species generated in the mitochondria. Manganese superoxide dismutase (SOD2) is the major enzymatic superoxide scavenger present in the mitochondrial matrix and one of the most crucial reactive oxygen species-scavenging enzymes in the cell. SOD2 is activated by sirtuin 3 (SIRT3) through NAD⁺-dependent deacetylation. However, the exact acetylation sites of SOD2 are ambiguous and the mechanisms underlying the deacetylation-mediated SOD2 activation largely remain unknown. We are the first to characterize SOD2 mutants of the acetylation sites by investigating the relative enzymatic activity, structures, and electrostatic potential of SOD2 in this study. These SOD2 mutations affected the superoxide-scavenging activity in vitro and in HEK293T cells. The lysine 68 (K68) site is the most important acetylation site contributing to SOD2 activation and plays a role in cell survival after paraquat treatment. The molecular basis underlying the regulation of SOD2 activity by K68 was investigated in detail. Molecular dynamics simulations revealed that K68 mutations induced a conformational shift of residues located in the active center of SOD2 and altered the charge distribution on the SOD2 surface. Thus, the entry of the superoxide anion into the coordinated core of SOD2 was inhibited. Our results provide a novel mechanistic insight, whereby SOD2 acetylation affects the structure and charge distribution of SOD2, its tetramerization, and p53–SOD2 interactions of SOD2 in the mitochondria, which may play a role in nuclear–mitochondrial communication during aging.     

The 2.0A resolution structure of the catalytic portion of a cyanobacterial membrane-bound manganese superoxide dismutase

Cyanobacteria are shown to be unique in containing membrane-bound manganese superoxide dismutases (MnSOD). They are homodimeric type 2 membrane proteins that protect this phototrophic organism against oxidative stress. We have determined, for the first time, the 2.0A resolution structure of the catalytic portion of the MnSOD from the filamentous cyanobacterium Anabaena PCC 7120. Within each subunit, both the N-terminal helical hairpin (His94 and His145) and the C-terminal alpha/beta domain (His232 and Asp228) contribute ligands to the catalytic manganese site. Together with a water or hydroxide ion (OH(x)) a five-coordinated trigonal bipyramidal geometry is formed, with OH(x) and His90 forming the axial ligands and manganese shifted out of the equatorial plane in the direction of OH(x). The ligands including OH(x) are tightly constrained by hydrogen bonding with surrounding residues either from the same monomer (Tyr98, Asn144, Trp194, Gln213, Val229, Trp230) or from the neighbouring subunit (Glu231, Tyr235). This underlines the important role of the symmetric dimeric structure of MnSODs in contributing elements to both the active site and the substrate funnel. The Mn cdots, three dots, centered Mn distance (18.4A) is bridged by the hydrogen-bonded His232 of one monomer with Glu231 of the other monomer. A detailed discussion of the structure, a comparison with known structures of soluble MnSODs as well as a model of the cyanobacterial membrane-bound MnSOD is presented.     

Comparison of the crystal structures of genetically engineered human manganese superoxide dismutase and manganese superoxide dismutase from Thermus thermophilus: differences in dimer-dimer interaction.

The three-dimensional X-ray structure of a recombinant human mitochondrial manganese superoxide dismutase (MnSOD) (chain length 198 residues) was determined by the method of molecular replacement using the related structure of MnSOD from Thermus thermophilus as a search model. This tetrameric human MnSOD crystallizes in space group P2(1)2(1)2 with a dimer in the asymmetric unit (Wagner, U.G., Werber, M.M., Beck, Y., Hartman, J.R., Frolow, F., & Sussman, J.L., 1989, J. Mol. Biol. 206, 787-788). Refinement of the protein structure (3,148 atoms with Mn and no solvents), with restraints maintaining noncrystallographic symmetry, converged at an R-factor of 0.207 using all data from 8.0 to 3.2 A resolution and group thermal parameters. The monomer-monomer interactions typical of bacterial Fe- and Mn-containing SODs are retained in the human enzyme, but the dimer-dimer interactions that form the tetramer are very different from those found in the structure of MnSOD from T. thermophilus. In human MnSOD one of the dimers is rotated by 84 degrees relative to its equivalent in the thermophile enzyme. As a result the monomers are arranged in an approximately tetrahedral array, the dimer-dimer packing is more intimate than observed in the bacterial MnSOD from T. thermophilus, and the dimers interdigitate. The metal-ligand interactions, determined by refinement and verified by computation of omit maps, are identical to those observed in T. thermophilus MnSOD.     

Three-dimensional location of ribosomal protein BL2 from Bacillus stearothermophilus, a key component of the peptidyl transferase center

Protein BL2 from Bacillus stearothermophilus has been localized by immunoelectron microscopy on the interface side of the 50 S subunit, beneath the angle formed between the central protuberance and the L1 protuberance. The immuno-electron microscopic data suggest that the interface region of the 50 S particle is not as flat as most of the proposed three-dimensional models suggest, but instead there is a significant concavity. Since several studies demonstrated that BL2 is implicated in peptidyl transferase activity or at least located close to the peptidyl transferase center, the location of protein BL2 also provides information as to the location of this important functional domain.     

Purification and properties of the manganese superoxide dismutase from the liver of bullfrog, Rana catesbeiana

A manganese superoxide dismutase (Mn-SOD) from the liver of bullfrog, Rana catesbeiana, was purified to electrophoretic homogeneity. The enzyme has a molecular weight of about 84,000 and is composed of four identical subunits, each containing one manganese atom. The amino acid composition of the enzyme is similar to that of Mn-SODs isolated from human and chicken livers, but differs considerably from that of the Escherichia coli enzyme (D. Barra et al. (1984) J. Biol. Chem. 259, 12595-12601; R. A. Weisiger and I. Fridovich (1973) J. Biol. Chem. 248, 3582-3592; H. M. Steinman (1978) J. Biol. Chem. 253, 8708-8720). The N-terminal amino acid is lysine. The sequence of 23 amino acid residues in the N-terminal region was determined. It shows excellent homologies with those of the human and chicken enzymes (H. M. Steinmam and R. L. Hill (1973) Proc. Natl. Acad. Sci. USA 70, 3725-3729; C. Ditlow et al. (1982) Carlsberg Res. Commun. 47, 81-91). The frog liver enzyme is also located exclusively in the mitochondrial matrix. Immunologically the same enzyme is also found in the tadpole liver, in an amount of about one-half of that in the adult bullfrog.     

Unusual stability of manganese superoxide dismutase from a new species, Tatumella ptyseos ct: its gene structure, expression, and enzyme properties

A genomic DNA of 1416 bp containing an open reading frame encoding a manganese superoxide dismutase (Mn-SOD) from Tatumella ptyseos ct was cloned. Sequence analysis of this new gene revealed that it translates 205 amino acid residues. The deduced amino acid sequence showed variable identities (41-91%) with sequences of Mn-SODs from other species. The residues required to coordinate the single trivalent manganese ion and the 11 residues putatively involved in the active center are conserved as they are in other reported Mn-SODs. In addition, the gene was introduced into the expression vector, pET-20b(+), and transformed in Escherichia coli BL21(DE3). The Mn-SOD was purified by a His-tag technique. The yield was 0.9 mg from 0.5 L of culture. The specific activity was 6540 U/mg. A dimer is the major form of the enzyme in equilibrium. The half-life of dimer is approximately 50 min and its thermal inactivation rate constant k(d) was 0.015 min(-1) at 80 degrees C. The dimerization of the enzyme was inhibited under an acidic pH (below 4.0), or in the presence of SDS (above 1%) or imidazole (above 0.5 M), whereas it was not affected under an alkaline pH (above 9.0). Furthermore, the dimeric enzyme was much more resistant to proteolytic attack after 3 h of incubation at 37 degrees C with trypsin and chymotrypsin. This unusually stable enzyme can be used as cosmetic to the protection of skin against the unaesthetic effects caused by free radicals.     

Conformational stability of recombinant manganese superoxide dismutase from the filamentous fungus Trichoderma reesei

Superoxide dismutases (SODs; EC 1.15.1.1) are part of the antioxidant system of aerobic organisms and are used as a defense against oxidative injury caused by reactive oxygen species (ROS). The cloning and sequencing of the 788-bp genomic DNA from Trichoderma reesei strain QM9414 (anamorph of Hypocrea jecorina) revealed an open reading frame encoding a protein of 212 amino acid residues, with 65-90% similarity to manganese superoxide dismutase from other filamentous fungi. The TrMnSOD was purified and shown to be stable from 20 to 90 °C for 1 h at pH from 8 to 11.5, while maintaining its biological activity.     

Cloning, sequencing, expression and characterization of the manganese superoxide dismutase gene from Vibrio alginolyticus.

The sodA gene coding for manganese superoxide dismutase from the marine microorganism Vibrio alginolyticus was cloned, sequenced and over-expressed in Escherichia coli using the pET20b (+) expression vector. The full-length gene was consisted of 603bp open reading frame, which encoded a polypeptide of 201 amino acid residues, with a calculated molecular weight of 22672Da. The deduced amino acid sequence of the sodA showed considerable homology to other Mn-SODs. The recombinant enzyme was efficiently purified from crude E. coli cell lysate by the metal ion affinity chromatography. The recombinant VAMn-SOD resisted thermo-denaturation up to 60 degrees C and was insensitive to inhibitors such as H2O2, NaN3 and diethyldithiocarbamic acid.     

Removing a hydrogen bond in the dimer interface of Escherichia coli manganese superoxide dismutase alters structure and reactivity

Among manganese superoxide dismutases, residues His30 and Tyr174 are highly conserved, forming part of the substrate access funnel in the active site. These two residues are structurally linked by a strong hydrogen bond between His30 NE2 from one subunit and Tyr174 OH from the other subunit of the dimer, forming an important element that bridges the dimer interface. Mutation of either His30 or Tyr174 in Escherichia coli MnSOD reduces the superoxide dismutase activity to 30--40% of that of the wt enzyme, which is surprising, since Y174 is quite remote from the active site metal center. The 2.2 A resolution X-ray structure of H30A-MnSOD shows that removing the Tyr174-->His30 hydrogen bond from the acceptor side results in a significant displacement of the main-chain segment containing the Y174 residue, with local rearrangement of the protein. The 1.35 A resolution structure of Y174F-MnSOD shows that disruption of the same hydrogen bond from the donor side has much greater consequences, with reorientation of F174 having a domino effect on the neighboring residues, resulting in a major rearrangement of the dimer interface and flipping of the His30 ring. Spectroscopic studies on H30A, H30N, and Y174F mutants show that (like the previously characterized Y34F mutant of E. coli MnSOD) all lack the high pH transition of the wt enzyme. This observation supports assignment of the pH sensitivity of MnSOD to coordination of hydroxide ion at high pH rather than to ionization of the phenolic group of Y34. Thus, mutations near the active site, as in the Y34F mutant, as well as at remote positions, as in Y174F, similarly affect the metal reactivity and alter the effective pK(a) for hydroxide ion binding. These results imply that hydrogen bonding of the H30 imidazole N--H group plays a key role in substrate binding and catalysis.     

Structure of Cu/Zn superoxide dismutase from the heavy-metal-tolerant yeast Cryptococcus liquefaciens strain N6

The deep-sea yeast Cryptococcus liquefaciens strain N6 shows high tolerance towards heavy metals, and can grow in the presence of high concentrations of copper ions. Enzymatic analysis indicated that copper ions induced the Cu/Zn superoxide dismutase activity of strain N6 (Cl-SOD1). In this study, the 1.2 Å resolution crystal structure of Cl-SOD1 has revealed several significant residue substitutions compared to the other Cu/Zn SODs. In the electrostatic loop, notably, His135 and Pro136 replace the well-conserved linear residues, while Thr133 substitutes a highly conserved glycine. The electrostatic loop has been shown to be involved in the copper uptake process, and these substitutions have caused an inward dragging of the turn region of the loop. As the introduction of proline and abolishment of glycine decrease loop flexibility, this structural reorganization may have helped stabilize the loop conformation, possibly resulting in more efficient copper uptake and a more stabilized copper-bound form.     

Crystal structure of auracyanin, a "blue" copper protein from the green thermophilic photosynthetic bacterium Chloroflexus aurantiacus

Auracyanin B, one of two similar blue copper proteins produced by the thermophilic green non-sulfur photosynthetic bacterium Chloroflexus aurantiacus, crystallizes in space group P6(4)22 (a=b=115.7 A, c=54.6 A). The structure was solved using multiple wavelength anomalous dispersion data recorded about the CuK absorption edge, and was refined at 1.55 A resolution. The molecular model comprises 139 amino acid residues, one Cu, 247 H(2)O molecules, one Cl(-) and two SO(4)(2-). The final residual and estimated standard uncertainties are R=0.198, ESU=0.076 A for atomic coordinates and ESU=0.05 A for Cu---ligand bond lengths, respectively. The auracyanin B molecule has a standard cupredoxin fold. With the exception of an additional N-terminal strand, the molecule is very similar to that of the bacterial cupredoxin, azurin. As in other cupredoxins, one of the Cu ligands lies on strand 4 of the polypeptide, and the other three lie along a large loop between strands 7 and 8. The Cu site geometry is discussed with reference to the amino acid spacing between the latter three ligands. The crystallographically characterized Cu-binding domain of auracyanin B is probably tethered to the periplasmic side of the cytoplasmic membrane by an N-terminal tail that exhibits significant sequence identity with known tethers in several other membrane-associated electron-transfer proteins.