Isolation of an active and heat-stable monomeric form of Cu,Zn superoxide dismutase from the periplasmic space of Escherichia coli (original) (raw)
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Archives of Biochemistry and Biophysics, 1997
Brucella abortus (8) and other Brucella species (9), The periplasmic Cu,Zn superoxide dismutase Haemophilus influenzae, H. parainfluenzae (10), and (Cu,ZnSOD) from Escherichia coli has been shown other Haemophilus species (11). It began to appear by sedimentation equilibrium to be a monomer with likely that Cu,ZnSODs were in fact widely distributed a molecular weight of Ç17,000. The enzyme suffered among gram-negative bacteria and this change of view a reversible inactivation when heated to 70ЊC. This gained impetus from the finding of Cu,ZnSOD in Eschewas minimized by added Cu(II) or Zn(II). Heat labilrichia coli (12), in Salmonella typhimurium (13), and ity was greater in phosphate than in Tris buffer. The in Legionella pneumophila (14). enzyme exhibited a time-dependent inactivation by In P. leiognathi (15), C. crescentis (16, 17), E. coli (12, Hg(II) and this too was greater in phosphate than 18), S. typhimurium (13), B. abortus (19), and in L. in Tris. This behavior can be explained by a modest pneumophila (20), the Cu,ZnSOD is periplasmic. It is affinity of the enzyme for Cu(II) and Zn(II) which not yet certain whether there are periplasmic sources results in a dissociation/association equilibrium. Elof O 0 2 , or whether this Cu,ZnSOD protects primarily evation of the temperature shifts this equilibrium against exogenous O 0 2. However, it is clear that this toward dissociation and phosphate sequesters the enzyme is induced late in the growth cycle (12, 21, 22), released metals making them less available for reinis important for survival in stationary phase (14), and, sertion at the active site. Hg(II) competes for occuin mutants lacking SodA and SodB, contributes to the pancy of the active site and there were more unoccuability to grow aerobically (23). Whether the Cu,ZnSOD pied sites in phosphate than in Tris. A parallel was functions as a pathogenicity factor in B. abortus has drawn between the E. coli Cu,ZnSOD and FALS vabeen suggested but is still unsettled (24, 25). rients of human Cu,ZnSOD, which are also relatively When first studied (12) the E. coli Cu,ZnSOD was unstable and exhibit low affinity for Cu(II). ᭧ 1997 found to be much less stable than the more thoroughly Academic Press characterized eukaryotic Cu,ZnSODs and, like them, was assumed to be dimeric. The subsequent report of a stable, monomeric Cu,ZnSOD from E. coli (26) indi-Copper-and zinc-containing superoxide dismucated that the stability and the quaternary structure tases (Cu,ZnSODs) 3 were thought to be restricted to of the E. coli Cu,ZnSOD would merit further study. We eukaryotes (1-4). This conventional wisdom was therefore used sedimentation equilibrium to establish called into question by sporadic reports of Cu,ZnSOD the molecular weight of this enzyme and examined in diverse bacteria. The first was from the marine those factors which influence its stability. The results bacterium Photobacter leiognathi (5). This was folof these and related studies are reported herein. lowed by descriptions of Cu,ZnSODs from Caulobacter crescentis (6), several species of pseudomonads MATERIALS AND METHODS Bacto-tryptone and yeast extract were from Difco and bovine 1 This work was supported by grants from the Council for Tobacco Cu,ZnSOD was from Diagnostic Data Inc. Xanthine oxidase was prepared from bovine cream by the procedure of Waud et al. (27). E. coli Research-U.S.A., Inc. (2871AR2) and the U. S. Army Medical Research Office (Contract DAMD17-95-C-5065). Cu,ZnSOD was prepared as previously described (28) from strain DH5a [supE44 DlacU169 (f80 lacZ Du15) hsd R17 recA endA 2 To whom correspondence should be addressed. Fax: (919) 684-8885. gyrA96 thi1 relA1] which overproduces the Cu,ZnSOD by virtue of the sod c containing plasmid psod C2.1 (22). This strain was kindly 3 Abbreviation used: Cu,ZnSODs, copper-and zinc-containing superoxide dismutases. provided by J. A. Imlay. Cells were grown to stationary phase in 305
Purification and partial characterization of Cu/Zn superoxide dismutase from
Journal of chromatography. B, Analytical technologies in the biomedical and life sciences
A new thermostable Cu/Zn SOD from a thermotolerant yeast strain Kluyveromyces marxianus NBIMCC 1984 has been purified and characterized. The purification procedure comprises thermal treatment and dialysis, ion-exchange chromatography and chromatofocusing. The methodology is a rapid, efficient and highly specific, generating pure preparation (specific activity 996 U mg of protein(-1)) with a yield of 53%. The purified enzyme is a homodimer with Mw of 34,034 Da and has high N-terminal homology with other yeasts' Cu/Zn SOD enzymes. The protein is characterized with some unique features such as-thermostability (t(1/2) at 70 degrees C=30 min), pH stability in the alkaline range (7.5-8.5) and resistance to inhibitors and variety of chemicals. These characteristics reveal possibilities for wide practical application of K. marxianus Cu/Zn SOD enzyme.
Journal of Molecular Biology, 1997
The ®rst three-dimensional structure of a functional monomeric Cu,Zn superoxide dismutase (from Escherichia coli, E SOD) is reported at 2.0 A Ê resolution (R-factor 16.8%). Compared to the homologous eukaryotic enzymes, E SOD displays a perturbed antiparallel b-barrel structure. The most striking structural features observed include extended amino acid insertions in the surface 1,2-loop and S-S subloop, modi®cation of the dis-ul®de bridge connection, and loss of functional electrostatic residues, suggesting a modi®ed 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.
Journal of Molecular Biology, 1997
The ®rst three-dimensional structure of a functional monomeric Cu,Zn superoxide dismutase (from Escherichia coli, E SOD) is reported at 2.0 A Ê resolution (R-factor 16.8%). Compared to the homologous eukaryotic enzymes, E SOD displays a perturbed antiparallel b-barrel structure. The most striking structural features observed include extended amino acid insertions in the surface 1,2-loop and S-S subloop, modi®cation of the dis-ul®de bridge connection, and loss of functional electrostatic residues, suggesting a modi®ed 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.
In vivo formation of Cu,Zn superoxide dismutase disulfide bond in Escherichia coli
FEBS Letters, 1999
We have found that the in vivo folding of periplasmic Escherichia coli Cu,Zn superoxide dismutase is assisted by DsbA, which catalyzes the efficient formation of its single disulfide bond, whose integrity is essential to ensure full catalytic activity to the enzyme. In line with these findings, we also report that the production of recombinant Xenopus laevis Cu,Zn superoxide dismutase is enhanced when the enzyme is exported in the periplasmic space or is expressed in thioredoxin reductase mutant strains. Our data show that inefficient disulfide bond oxidation in the bacterial cytoplasm inhibits Cu,Zn superoxide dismutase folding in this cellular compartment.
Role of the Dimeric Structure in Cu,Zn Superoxide Dismutase
Journal of Biological Chemistry, 1998
To investigate the structural/functional role of the dimeric structure in Cu,Zn superoxide dismutases, we have studied the stability to a variety of agents of the Escherichia coli enzyme, the only monomeric variant of this class so far isolated. Differential scanning calorimetry of the native enzyme showed the presence of two well defined peaks identified as the metal free and holoprotein. Unlike dimeric Cu,Zn superoxide dismutases, the unfolding of the monomeric enzyme was found to be highly reversible, a behavior that may be explained by the absence of free cysteines and the highly polar nature of its molecular surface. The melting temperature of the E. coli enzyme was found to be pH-dependent with the holoenzyme transition centered at 66°C at pH 7.8 and at 79.3°C at pH 6.0. The active-site metals, which were easily displaced from the active site by EDTA, were found to enhance the thermal stability of the monomeric apoprotein but to a lower extent than in the dimeric enzymes from eukaryotic sources. Apo-superoxide dismutase from E. coli was shown to be nearly as stable as the bovine apoenzyme, whose holo form is much more stable and less sensitive to pH variations. The remarkable pH susceptibility of the E. coli enzyme structure was paralleled by the slow decrease in activity of the enzyme incubated at alkaline pH and by modification of the EPR spectrum at lower pH values than in the case of dimeric enzymes. Unlike eukaryotic Cu,Zn superoxide dismutases, the active-site structure of the E. coli enzyme was shown to be reversibly perturbed by urea. These observations suggest that the conformational stability of Cu,Zn superoxide dismutases is largely due to the intrinsic stability of the -barrel fold rather than to the dimeric structure and that pH sensitivity and weak metal binding of the E. coli enzyme are due to higher flexibility and accessibility to the solvent of its activesite region.
Febs Letters, 2000
The active site of the Cu,Zn superoxide dismutase from Escherichia coli in the oxidized Cu(II) state has been studied by nuclear magnetic relaxation dispersion (NMRD), optical and nuclear magnetic resonance spectroscopy. The orientation of some metal ligands is different with respect to all the other Cu,Zn superoxide dismutases. Moreover, NMRD measurements demonstrate the lack of a copper-coordinated water molecule. In spite of these differences the enzymatic activity is still high. Azide also binds copper with normal affinity and induces modifications in the active site comparable to those previously observed in the eukaryotic enzymes. Our results suggest that, in this enzyme, the copper-coordinated water molecule appears not necessary for the enzymatic reaction. A role for the copper-coordinated water molecule is discussed in the light of recent crystallographic studies. ß
Proceedings of the National Academy of Sciences, 2005
Little is known about prokaryotic homologs of Cu,Zn superoxide dismutase (SOD), an enzyme highly conserved among eukaryotic species. In 138 Archaea and Bacteria genomes, 57 of these putative homologs were found, 11 of which lack at least one of the metal ligands. Both the solution and the crystal structures of the SOD-like protein from Bacillus subtilis, lacking two Cu ligands and found to be enzymatically inactive, were determined. In solution, the protein is monomeric. The available nuclear Overhauser effects, together with chemical-shift index values, allowed us to define and to recognize the typical Cu,Zn SOD Greek -barrel but with largely unstructured loops (which, therefore, sample a wide range of conformations).