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>