10.1371/journal.pone.0099492 Diogo de Abreu Meireles Diogo de Abreu Meireles Thiago Geronimo Pires Alegria Thiago Geronimo Pires Alegria Simone Vidigal Alves Simone Vidigal Alves Carla Rani Rocha Arantes Carla Rani Rocha Arantes Luis Eduardo Soares Netto Luis Eduardo Soares Netto A 14.7 kDa Protein from <i>Francisella tularensis subsp. novicida</i> (Named FTN_1133), Involved in the Response to Oxidative Stress Induced by Organic Peroxides, Is Not Endowed with Thiol-Dependent Peroxidase Activity Public Library of Science 2014 Biochemistry enzymology enzymes oxidoreductases Enzyme structure proteins Recombinant proteins chemical biology Protein chemistry microbiology Medical microbiology Microbial pathogens Bacterial pathogens Microbial physiology Pathology and laboratory medicine pathogenesis kda tularensis oxidative induced endowed thiol-dependent peroxidase 2014-06-24 03:21:48 Dataset 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 <div><p><i>Francisella</i> 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 <i>F. tularensis subsp. novicida</i>, 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 <i>in silico</i> prediction of an all-α-helix secondary structure. The pK<sub>a</sub> 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 <i>FTN_1133</i> gene in <i>ahpC</i> and <i>oxyR</i> mutants of <i>E. coli</i> showed no complementation. Furthermore, analysis of <i>FTN_1133</i> 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.</p></div>