%0 Generic %A de Abreu Meireles, Diogo %A Geronimo Pires Alegria, Thiago %A Vidigal Alves, Simone %A Rani Rocha Arantes, Carla %A Eduardo Soares Netto, Luis %D 2014 %T A 14.7 kDa Protein from Francisella tularensis subsp. novicida (Named FTN_1133), Involved in the Response to Oxidative Stress Induced by Organic Peroxides, Is Not Endowed with Thiol-Dependent Peroxidase Activity %U https://plos.figshare.com/articles/dataset/A_14_7_kDa_Protein_from_Francisella_tularensis_subsp_novicida_Named_FTN_1133_Involved_in_the_Response_to_Oxidative_Stress_Induced_by_Organic_Peroxides_Is_Not_Endowed_with_Thiol_Dependent_Peroxidase_Activity/1081452 %R 10.1371/journal.pone.0099492 %2 https://plos.figshare.com/ndownloader/files/1562488 %2 https://plos.figshare.com/ndownloader/files/1562489 %2 https://plos.figshare.com/ndownloader/files/1562490 %2 https://plos.figshare.com/ndownloader/files/1562491 %K Biochemistry %K enzymology %K enzymes %K oxidoreductases %K Enzyme structure %K proteins %K Recombinant proteins %K chemical biology %K Protein chemistry %K microbiology %K Medical microbiology %K Microbial pathogens %K Bacterial pathogens %K Microbial physiology %K Pathology and laboratory medicine %K pathogenesis %K kda %K tularensis %K oxidative %K induced %K endowed %K thiol-dependent %K peroxidase %X

Francisella genus comprises Gram-negative facultative intracellular bacteria that are among the most infectious human pathogens. A protein of 14.7 KDa named as FTN_1133 was previously described as a novel hydroperoxide resistance protein in F. tularensis subsp. novicida, implicated in organic peroxide detoxification and virulence. Here, we describe a structural and biochemical characterization of FTN_1133. Contrary to previous assumptions, multiple amino acid sequence alignment analyses revealed that FTN_1133 does not share significant similarity with proteins of the Ohr/OsmC family or any other Cys-based, thiol dependent peroxidase, including conserved motifs around reactive cysteine residues. Circular dichroism analyses were consistent with the in silico prediction of an all-α-helix secondary structure. The pKa of its single cysteine residue, determined by a monobromobimane alkylation method, was shown to be 8.0±0.1, value that is elevated when compared with other Cys-based peroxidases, such as peroxiredoxins and Ohr/OsmC proteins. Attempts to determine a thiol peroxidase activity for FTN_1133 failed, using both dithiols (DTT, thioredoxin and lipoamide) and monothiols (glutathione or 2-mercaptoethanol) as reducing agents. Heterologous expression of FTN_1133 gene in ahpC and oxyR mutants of E. coli showed no complementation. Furthermore, analysis of FTN_1133 protein by non-reducing SDS-PAGE showed that an inter-molecular disulfide bond (not detected in Ohr proteins) can be generated under hydroperoxide treatment, but the observed rates were not comparable to those observed for other thiol-dependent peroxidases. All the biochemical and structural data taken together indicated that FTN_1133 displayed distinct characteristics from other thiol dependent peroxidases and, therefore, suggested that FTN_1133 is not directly involved in hydroperoxide detoxification.

%I PLOS ONE