Functional and crystallographic characterization of< i> Salmonella typhimurium Cu, Zn superoxide dismutase coded by the< i> sodCI virulence gene (original) (raw)
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Journal of Molecular Biology, 2000
The functional and three-dimensional structural features of Cu,Zn superoxide dismutase coded by the Salmonella typhimurium sodCI gene, have been characterized. Measurements of the catalytic rate indicate that this enzyme is the most ef®cient superoxide dismutase analyzed so far, a feature that may be related to the exclusive association of the sodCI gene with the most pathogenic Salmonella serotypes. The enzyme active-site copper ion is highly accessible to external probes, as indicated by quenching of the water proton relaxation rate upon addition of iodide. The shape of the electron paramagnetic resonance spectrum is dependent on the frozen or liquid state of the enzyme solution, suggesting relative¯exibility of the copper ion environment. The crystal structure (R-factor 22.6 %, at 2.3 A Ê resolution) indicates that the dimeric enzyme adopts the quaternary assembly typical of prokaryotic Cu,Zn superoxide dismutases. However, when compared to the structures of the homologous enzymes from Photobacterium leiognathi and Actinobacillus pleuropneumoniae, the subunit interface of Salmonella Cu,Zn superoxide dismutase shows substitution of 11 out of 19 interface residues. As a consequence, the network of structural water molecules that ®ll the dimer interface cavity is structured differently from the other dimeric bacterial enzymes. The crystallographic and functional characterization of this Salmonella Cu,Zn superoxide dismutase indicates that structural variability and catalytic ef®ciency are higher in prokaryotic than in the eukaryotic homologous enzymes.
Journal of Molecular Biology, 2000
The functional and three-dimensional structural features of Cu,Zn superoxide dismutase coded by the Salmonella typhimurium sodCI gene, have been characterized. Measurements of the catalytic rate indicate that this enzyme is the most ef®cient superoxide dismutase analyzed so far, a feature that may be related to the exclusive association of the sodCI gene with the most pathogenic Salmonella serotypes. The enzyme active-site copper ion is highly accessible to external probes, as indicated by quenching of the water proton relaxation rate upon addition of iodide. The shape of the electron paramagnetic resonance spectrum is dependent on the frozen or liquid state of the enzyme solution, suggesting relative¯exibility of the copper ion environment. The crystal structure (R-factor 22.6 %, at 2.3 A Ê resolution) indicates that the dimeric enzyme adopts the quaternary assembly typical of prokaryotic Cu,Zn superoxide dismutases. However, when compared to the structures of the homologous enzymes from Photobacterium leiognathi and Actinobacillus pleuropneumoniae, the subunit interface of Salmonella Cu,Zn superoxide dismutase shows substitution of 11 out of 19 interface residues. As a consequence, the network of structural water molecules that ®ll the dimer interface cavity is structured differently from the other dimeric bacterial enzymes. The crystallographic and functional characterization of this Salmonella Cu,Zn superoxide dismutase indicates that structural variability and catalytic ef®ciency are higher in prokaryotic than in the eukaryotic homologous enzymes.
Biochemistry, 2008
The structure of the SodCII-encoded monomeric Cu, Zn superoxide dismutase from Salmonella enterica has been solved by NMR spectroscopy. This represents the first solution structure of a natural and fully active monomeric superoxide dismutase in solution, providing information useful for the interpretation of the evolutional development of these enzymes. The protein scaffold consists of the characteristic -barrel common to the whole enzyme family. The general shape of the protein is quite similar to that of Escherichia coli Cu, Zn superoxide dismutase, although some differences are observed mainly in the active site. SodCII presents a more rigid conformation with respect to the engineered monomeric mutants of the human Cu, Zn superoxide dismutase, even though significant disorder is still present in the loops shaping the active site. The analysis of both dynamics and hydration properties of the protein in solution highlights the factors required to maintain the fully active and, at the same time, monomeric protein. This study provides novel insights into the functional differences between monomeric and dimeric bacterial Cu, Zn superoxide dismutases, in turn helping to explain the convergent evolution toward a dimeric structure in prokaryotic and eukaryotic enzymes of this class. † CIRMMP is gratefully acknowledged for providing access to the high field instrumentation available at CERM, Florence
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, 2000
Macrophages and neutrophils protect animals from microbial infection in part by issuing a burst of toxic superoxide radicals when challenged. To counteract this onslaught, many Gram-negative bacterial pathogens possess periplasmic Cu,Zn superoxide dismutases (SODs), which act on superoxide to yield molecular oxygen and hydrogen peroxide. We have solved the X-ray crystal structure of the Cu,Zn SOD from Actinobacillus pleuropneumoniae, a major porcine pathogen, by molecular replacement at 1.9 A Ê resolution. The structure reveals that the dimeric bacterial enzymes form a structurally homologous class de®ned by a water-mediated dimer interface, and share with all Cu,Zn SODs the Greek-key b-barrel subunit fold with copper and zinc ions located at the base of a deep loopenclosed active-site channel. Our structure-based sequence alignment of the bacterial enzymes explains the monomeric nature of at least two of these, and suggests that there may be at least one additional structural class for the bacterial SODs. Two metal-mediated crystal contacts yielded our C222 1 crystals, and the geometry of these sites could be engineered into proteins recalcitrant to crystallization in their native form. This work highlights structural differences between eukaryotic and prokaryotic Cu,Zn SODs, as well as similarities and differences among prokaryotic SODs, and lays the groundwork for development of antimicrobial drugs that speci®cally target periplasmic Cu,Zn SODs of bacterial pathogens.
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.
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).