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

Author(s)

Meireles, Diogo de Abreu ^[1] ; Pires Alegria, Thiago Geronimo ^[1] ; Alves, Simone Vidigal ^[1] ; Rocha Arantes, Carla Rani ^[1] ; Soares Netto, Luis Eduardo ^[1]

Affiliation

^[1] Univ Sao Paulo, Inst Biociencias, Dept Genet & Biol Evolut, Sao Paulo - Brazil

• Document type: Journal article
• Source: PLoS One; v. 9, n. 6 JUN 24 2014.
• Web of Science Citations: 5

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-a-helix secondary structure. The pK(a) 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 intermolecular 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. (AU)

• FAPESP's process: 13/07937-8 - Redoxome - Redox Processes in Biomedicine
• Grantee: Ohara Augusto
• Support type: Research Grants - Research, Innovation and Dissemination Centers - RIDC
• FAPESP's process: 12/21722-1 - Structural and biochemical characterization of Ohr/OsmC family proteins: emphasis on the characterization of a Francisella tularensis protein involved in pathogenicity
• Grantee: Diogo de Abreu Meireles
• Support type: Scholarships in Brazil - Post-Doctorate
• FAPESP's process: 07/58147-6 - Biological aspects of thiols: protein structure, antioxidant defense, cell signaling and redox states
• Grantee: Luis Eduardo Soares Netto
• Support type: Research Projects - Thematic Grants