Vibrio cholerae periplasmic superoxide dismutase: Isolation of the gene and overexpression of the protein
University of Rome Tor Vergata, Roma, Latium, Italy Journal of Biotechnology
(Impact Factor: 2.87).
05/2004; 109(1-2):123-30. DOI: 10.1016/j.jbiotec.2004.01.002
Superoxide dismutases are ubiquitous enzymes which play an important role in protecting cells against oxidative damage and which have also been shown to contribute to the pathogenicity of many bacterial species. Here we demonstrate that Vibrio cholerae, the causative agent of cholerae, expresses an active periplasmic Cu,Zn superoxide dismutase. Moreover, we have set up an expression system yielding large amounts of V. cholerae recombinant Cu,Zn superoxide dismutase in the periplasm of Escherichia coli and a procedure to obtain the enzyme in a highly purified form. Unlike the bovine enzyme, V. cholerae Cu,Zn superoxide dismutase has been proved to be highly resistant to inactivation by hydrogen peroxide. This property, which appears to be common to other bacterial enzymes of this class, might improve the ability of Cu,Zn superoxide dismutase to protect bacteria against the reactive oxygen species produced by phagocytes.
Available from: Andrea Battistoni
- "Signal peptidase I (SpI) removes the leader sequence from proteins which are to be exported in the periplasmic space or secreted in the extracellular milieu, while signal peptidase II (SpII) removes the leader peptide from lipid-modified precursor of exported proteins, which are targeted to the periplasmic inner and outer membranes. The N-terminal region of most SodC proteins from Gram-negative bacteria shows the typical features of the signal peptides recognized by SpI , whereas the signal sequences of mycobacterial SodC proteins are recognized by SpII . Analysis of the amino acids sequences of SodC-F1 and SodC-F2 through the Lipo P program showed that the leader peptide sequence of the two prophage encoded sodC genes possess features compatible with processing either by SpI or by SpII (Figure 3). "
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ABSTRACT: Highly virulent enterohemorrhagic Escherichia coli O157:H7 strains possess three sodC genes encoding for periplasmic Cu, Zn superoxide dismutases: sodC, which is identical to the gene present in non-pathogenic E. coli strains, and sodC-F1 and sodC-F2, two nearly identical genes located within lambdoid prophage sequences. The significance of this apparent sodC redundancy in E. coli O157:H7 has not yet been investigated.
We report that strains deleted of one or more sodC genes are less resistant than the wild type strain to a challenge with hydrogen peroxide, thus confirming their involvement in the bacterial antioxidant apparatus. To understand if the different sodC genes have truly overlapping functions, we have carried out a comparison of the functional, structural and regulatory properties of the various E. coli O157:H7 SodC enzymes. We have found that the chromosomal and prophagic sodC genes are differentially regulated in vitro. sodC is exclusively expressed in aerobic cultures grown to the stationary phase. In contrast, sodC-F1 and sodC-F2 are expressed also in the logarithmic phase and in anaerobic cultures. Moreover, the abundance of SodC-F1/SodC-F2 increases with respect to that of SodC in bacteria recovered from infected Caco-2 cells, suggesting higher expression/stability of SodC-F1/SodC-F2 in intracellular environments. This observation correlates with the properties of the proteins. In fact, monomeric SodC and dimeric SodC-F1/SodC-F2 are characterized by sharp differences in catalytic activity, metal affinity, protease resistance and stability.
Our data show that the chromosomal and bacteriophage-associated E. coli O157:H7 sodC genes have different regulatory properties and encode for proteins with distinct structural/functional features, suggesting that they likely play distinctive roles in bacterial protection from reactive oxygen species. In particular, dimeric SodC-F1 and SodC-F2 possess physico-chemical properties which make these enzymes more suitable than SodC to resist the harsh environmental conditions which are encountered by bacteria within the infected host.
BMC Microbiology 11/2008; 8(1):166. DOI:10.1186/1471-2180-8-166 · 2.73 Impact Factor
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ABSTRACT: Bacterial and eukaryotic Cu,Zn superoxide dismutases show remarkable differences in the active site region and in their quaternary structure organization. We report here a functional comparison between four Cu,Zn superoxide dismutases from Gram-negative bacteria and the eukaryotic bovine enzyme. Our data indicate that bacterial dimeric variants are characterized by catalytic rates higher than that of the bovine enzyme, probably due to the solvent accessibility of their active site. Prokaryotic Cu,Zn superoxide dismutases also show higher resistance to hydrogen peroxide inactivation and lower HCO3- -dependent peroxidative activity. Moreover, unlike the eukaryotic enzyme, all bacterial variants are susceptible to inactivation by chelating agents and show variable sensitivity to proteolytic attack, with the E. coli monomeric enzyme showing higher rates of inactivation by EDTA and proteinase K. We suggest that differences between individual bacterial variants could be due to the influence of modifications at the dimer interface on the enzyme conformational flexibility.
Biological Chemistry 09/2004; 385(8):749-54. DOI:10.1515/BC.2004.091 · 3.27 Impact Factor
Available from: brandeis.edu
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ABSTRACT: Copper-zinc superoxide dismutase (CuZnSOD, SOD1 protein) is an abundant copper- and zinc-containing protein that is present in the cytosol, nucleus, peroxisomes, and mitochondrial intermembrane space of human cells. Its primary function is to act as an antioxidant enzyme, lowering the steady-state concentration of superoxide, but when mutated, it can also cause disease. Over 100 different mutations have been identified in the sod1 genes of patients diagnosed with the familial form of amyotrophic lateral sclerosis (fALS). These mutations result in a highly diverse group of mutant proteins, some of them very similar to and others enormously different from wild-type SOD1. Despite their differences in properties, each member of this diverse set of mutant proteins causes the same clinical disease, presenting a challenge in formulating hypotheses as to what causes SOD1-associated fALS. In this review, we draw together and summarize information from many laboratories about the characteristics of the individual mutant SOD1 proteins in vivo and in vitro in the hope that it will aid investigators in their search for the cause(s) of SOD1-associated fALS.
Annual Review of Biochemistry 02/2005; 74(1):563-93. DOI:10.1146/annurev.biochem.72.121801.161647 · 30.28 Impact Factor
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