Meireles Diogo de Abreu, Alegria Thiago Geronimo Pires, Alves Simone Vidigal, Arantes Carla Rani Rocha, Netto Luis Eduardo Soares
Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, São Paulo, Brazil.
PLoS One. 2014 Jun 24;9(6):e99492. doi: 10.1371/journal.pone.0099492. eCollection 2014.
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.
弗朗西斯菌属包含革兰氏阴性兼性细胞内细菌，是最具感染力的人类病原体之一。一种名为FTN_1133的14.7千道尔顿蛋白质先前被描述为土拉热弗朗西斯菌新凶手亚种中的一种新型抗氢过氧化物蛋白，与有机过氧化物解毒和毒力有关。在此，我们描述了FTN_1133的结构和生化特征。与先前的假设相反，多氨基酸序列比对分析表明，FTN_1133与Ohr/OsmC家族的蛋白质或任何其他基于半胱氨酸的硫醇依赖性过氧化物酶没有显著相似性，包括反应性半胱氨酸残基周围的保守基序。圆二色性分析与全α螺旋二级结构的计算机预测一致。通过单溴代联苯胺烷基化方法测定，其单个半胱氨酸残基的pKa为8.0±0.1，与其他基于半胱氨酸的过氧化物酶（如过氧化物还原酶和Ohr/OsmC蛋白）相比，该值有所升高。使用二硫醇（二硫苏糖醇、硫氧还蛋白和硫辛酰胺）和单硫醇（谷胱甘肽或2-巯基乙醇）作为还原剂，测定FTN_1133硫醇过氧化物酶活性的尝试均失败。FTN_1133基因在大肠杆菌的ahpC和oxyR突变体中的异源表达未显示互补作用。此外，通过非还原SDS-PAGE分析FTN_1133蛋白表明，在氢过氧化物处理下可产生分子间二硫键（在Ohr蛋白中未检测到），但观察到的速率与其他硫醇依赖性过氧化物酶观察到的速率不可比。综合所有生化和结构数据表明，FTN_1133与其他硫醇依赖性过氧化物酶具有不同的特征，因此表明FTN_1133不直接参与氢过氧化物解毒。
