title: 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.
abstract: 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.
authors: Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, São Paulo, Brazil.
authors: Diogo de Abreu
authors: Dde A
authors: Meireles
authors: Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, São Paulo, Brazil.
authors: Thiago Geronimo Pires
authors: TG
authors: Alegria
authors: Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, São Paulo, Brazil.
authors: Simone Vidigal
authors: SV
authors: Alves
authors: Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, São Paulo, Brazil.
authors: Carla Rani Rocha
authors: CR
authors: Arantes
authors: Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, São Paulo, Brazil.
authors: Luis Eduardo Soares
authors: LE
authors: Netto
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D002942:Circular Dichroism; D017124:Conserved Sequence; D005604:Francisella tularensis / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D008958:Models, Molecular; D016877:Oxidants / Q000494:pharmacology*; D018384:Oxidative Stress; D010544:Peroxidases / Q000737:chemistry* / Q000378:metabolism*; D017433:Protein Structure, Secondary
chemical_list: D001426:Bacterial Proteins; D016877:Oxidants; D006861:Hydrogen Peroxide; D010544:Peroxidases