Functional and crystallographic characterization of Salmonella typhimurium Cu,Zn superoxide dismutase coded by the sodCI virulence gene 1 1Edited by R. Huber

Pesce, Alessandra; Battistoni, Andrea; Stroppolo, Maria Elena; Polizio, Francesca; Nardini, M.; Kroll, J. Simon; Langford, Paul R.; O’Neill, Peter; Sette, Marco; Desideri, Alessandro; Bolognesi, Martino

doi:10.1006/jmbi.2000.4074

Cited by 46 publications

(68 citation statements)

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“…Therefore, activity was standardized relative to the copper content of the sample. Purified SodCI displayed an activity of 18,600 units/mg, whereas SodCII showed an activity of 6,800 units/mg, confirming that SodCI possesses a very high catalytic rate (27).…”

Section: Contributions Of Sodci and Sodcii To Salmonellamentioning

confidence: 82%

“…Protein Purification-Recombinant SodCI was purified as described previously (27). SodCII was purified from E. coli 71/18 cells bearing plasmid pSEsodCII, obtained by inserting the sodCII gene in plasmid pSE420 under control of the trc promoter.…”

Section: Methodsmentioning

confidence: 99%

“…It was proposed that this phenomenon, described as SodCI tethering, might account for the greater contribution of this enzyme to Salmonella virulence. However, others have suggested that SodCI can be easily released from the periplasm when expressed in E. coli (27,29) or in Salmonella serovars other than typhimurium (16).…”

Section: Contributions Of Sodci and Sodcii To Salmonellamentioning

confidence: 99%

“…Although these proteins catalyze the same reaction, two groups have reported that the sodCI and sodCII genes are not interchangeable, suggesting that the two proteins have distinctive structural or functional properties (23,24). SodCI exhibits the highest catalytic rate ever observed in a natural Cu,Zn-SOD (27), but Cu,Zn-SODs in general are characterized by very high catalytic rates (28,29). More significantly, SodCI is a dimeric protein, whereas SodCII is a monomer (16).…”

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Regulatory and Structural Differences in the Cu,Zn-Superoxide Dismutases of Salmonella enterica and Their Significance for Virulence

Ammendola

Pasquali

Pacello

et al. 2008

Journal of Biological Chemistry

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Many of the most virulent strains of Salmonella enterica produce two distinct Cu,Zn-superoxide dismutases (SodCI and SodCII). The bacteriophage-encoded SodCI enzyme makes the greater contribution to Salmonella virulence. We have performed a detailed comparison of the functional, structural, and regulatory properties of the Salmonella SodC enzymes. Here we demonstrate that SodCI and SodCII differ with regard to specific activity, protease resistance, metal affinity, and peroxidative activity, with dimeric SodCI exhibiting superior stability and activity. In particular, monomeric SodCII is unable to retain its catalytic copper ion in the absence of zinc. We have also found that SodCI and SodCII are differentially affected by oxygen, zinc availability, and the transcriptional regulator FNR. SodCII is strongly down-regulated under anaerobic conditions and dependent on the high affinity ZnuABC zinc transport system, whereas SodCI accumulation in vitro and within macrophages is FNR-dependent. We have confirmed earlier findings that SodCII accumulation in intracellular Salmonella is negligible, whereas SodCI is strongly up-regulated in macrophages. Our observations demonstrate that differences in expression, activity, and stability help to account for the unique contribution of the bacteriophage-encoded SodCI enzyme to Salmonella virulence.

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Section: Contributions Of Sodci and Sodcii To Salmonellamentioning

confidence: 82%

Section: Methodsmentioning

confidence: 99%

Section: Contributions Of Sodci and Sodcii To Salmonellamentioning

confidence: 99%

mentioning

confidence: 99%

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Regulatory and Structural Differences in the Cu,Zn-Superoxide Dismutases of Salmonella enterica and Their Significance for Virulence

Ammendola

Pasquali

Pacello

et al. 2008

Journal of Biological Chemistry

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“…SOD has been considered for a long time to be peculiar of eukaryotic organisms, in which its evolutionary rate has been studied (5). From the first description of a SOD activity in Escherichia coli (6), other putative homologous of the eukaryotic protein have been then found in the periplasmic space of other bacterial species (7) The x-ray structures have been solved for the proteins from Photobacterium leiognathi (8), E. coli (9), Salmonella typhimurium (10), and Actinobacillus pleuropneumoniae (11), all of them maintaining the typical metal sites of Cu,Zn eukaryotic SODs and normal SOD activity. With the aim of providing a more detailed picture of prokaryotic SODs, we browsed the available complete genomes of Archea and Bacteria to search for sequences with some degree of homology with human SOD (HSOD), which is found in 48 of the 138 prokaryotes with complete genome.…”

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confidence: 99%

A prokaryotic superoxide dismutase paralog lacking two Cu ligands: From largely unstructured in solution to ordered in the crystal

Banci

Bertini

Calderone

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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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). On the contrary, in the crystal structure (obtained in the presence of slight excess of Zn), the protein is well structured and organized in covalent dimers held by a symmetric bridge consisting of a Zn ion bound to an Asp-His dyad in a tetrahedral geometry. Couples of dimers held by hydrophobic interactions and H bonds are further organized in long chains. The order͞disorder transition is discussed in terms of metal binding and physical state.conformational mobility ͉ NMR structure determination ͉ Zn-mediated protein dimers ͉ x-ray crystallography ͉ superoxide dismutase T he availability of the complete sequence of an increasing number of genomes allows comparative analyses aimed at the identification of genes and of corresponding physiological functions conserved along evolution. Cu,Zn superoxide dismutase (SOD) is a ubiquitous family of enzymes that efficiently catalyze the dismutation of superoxide anions into oxygen and hydrogen peroxide (1, 2). The protein takes a classical Greek key ␤-barrel fold and adopts a dimeric quaternary structure (3). It binds one Zn and one Cu ion per subunit, the latter ion being the site of the enzymatic reaction, whose rate limiting step is the diffusion of superoxide through the channel that leads to the Cu site (4). SOD has been considered for a long time to be peculiar of eukaryotic organisms, in which its evolutionary rate has been studied (5). From the first description of a SOD activity in Escherichia coli (6), other putative homologous of the eukaryotic protein have been then found in the periplasmic space of other bacterial species (7) The x-ray structures have been solved for the proteins from Photobacterium leiognathi (8), E. coli (9), Salmonella typhimurium (10), and Actinobacillus pleuropneumoniae (11), all of them maintaining the typical metal sites of Cu,Zn eukaryotic SODs and normal SOD activity. With the aim of providing a more detailed picture of prokaryotic SODs, we browsed the available complete genomes of Archea and Bacteria to search for sequences with some degree of homology with human SOD (HSOD), which is found in 48 of the 138 prokaryotes with complete genome. In some of them, not all of the metal ligands are present. The SOD-like protein from Bacillus subtilis (BsSOD) lacks two binding-Cu residues (His-48 and His-63, human protein numbe...

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Cocatalytic Zinc Sites

Auld¹

2004

Handbook of Metalloproteins

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Cocatalytic zinc sites occur in enzymes where two or three metals are closely grouped to bring about catalysis. There are over four dozen representatives of this type of zinc site with the great majority belonging to the Class III hydrolases. A novel feature of these sites is the bridging of two of the metal sites by a side chain moiety of a single amino acid residue, such as the ring nitrogens of the imidazole group of histidine or the carboxylate oxygens of aspartic acid, glutamic acid, or of a carboxylated lysine, LysCO 2 − . Such an interaction requires the metals to be in close proximity to each other. The ligands to cocatalytic zinc sites often come from nearly the entire length of the protein, indicating that the metal sites may be important to the overall fold of the protein as well as to catalytic function. The secondary structure of the protein plays a major role in providing the ligands to these sites. In most cases, ligands are provided by amino acids residing within one or two residues before or after a β‐sheet or an α‐helix. The zinc ions are often penta‐coordinate and arranged in a trigonal bipyramidal geometry. The bridging amino acids and H 2 O probably have critical roles in catalysis. Their dissociation from either metal atom during catalysis will change the charge on the metal promoting its action as a Lewis acid or allowing interaction with an electronegative atom of the substrate. Thus, it can be envisioned for hydrolytic enzymes that substrate binding involves one zinc site acting as a template for substrate binding, while the other zinc site provides hydroxide for nucleophilic attack on the sp 2 center of the ester or amide bond of the substrate. In the next step, the roles of the metals can be reversed. In this manner, the metal atoms and their associated ligands play specific roles in each step of the reaction that works to bring about catalysis. The ligands in these sites, in particular the histidines, are often involved in further hydrogen‐bonding interactions with other amino acids. These interactions should effect the charge on the metal and the stability of the metal complex, thus fine‐tuning catalysis and the stability of the metal sites.

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Functional and crystallographic characterization of Salmonella typhimurium Cu,Zn superoxide dismutase coded by the sodCI virulence gene 1 1Edited by R. Huber

Cited by 46 publications

References 56 publications

Regulatory and Structural Differences in the Cu,Zn-Superoxide Dismutases of Salmonella enterica and Their Significance for Virulence

Regulatory and Structural Differences in the Cu,Zn-Superoxide Dismutases of Salmonella enterica and Their Significance for Virulence

A prokaryotic superoxide dismutase paralog lacking two Cu ligands: From largely unstructured in solution to ordered in the crystal

Cocatalytic Zinc Sites

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