几种生物活性取决于铁硫簇([Fe-S])。尽管它们在几种生物体中是众所周知的,但在大多数生物体中对它们的功能和代谢途径知之甚少。我们建议使用阿米巴盘网柄菌,作为研究[Fe-S]在分子上生物合成的生物模型,细胞和生物体水平。首先,我们探索了D.discoideum基因组,寻找与构成Fe-S簇组装分子机制的亚基相对应的基因,基于哺乳动物超复合物的结构和氨基酸保守谱,我们推断了变形虫机械的全部功能。之后,我们表达了D.盘盘菌共轴蛋白(DdFXN)的重组成熟形式,该途径的动力学激活剂。我们表征了蛋白质及其构象稳定性。DdFXN是单体且致密的。二级结构内容的分析,使用远紫外CD光谱计算,与FXN折叠的预期数据兼容,和近UVCD光谱与对应于折叠蛋白质的数据兼容。此外,色氨酸荧光表明发射来自非极性环境。然而,DdFXN的构象明显不如人类FXN的构象稳定,(4.0vs.9.0kcalmol-1)。基于DdFXN的序列分析和结构模型,我们研究了参与DdFXN与超复合物相互作用的关键残基,以及点突变对DdFXN三级结构能量学的影响。Friedreich的共济失调中涉及的10多个残基在人和DdFXN形式之间保守,并发现两种蛋白质的能量突变效应之间存在良好的相关性,表明存在相似的序列/函数/稳定性关系。最后,我们将这些信息整合在一个进化背景中,该背景突出了变形虫和人类之间的特定变异模式,这可能反映了特定蛋白质位置的功能重要性。此外,获得的完整途径构成了支持Human和D.discoideum之间共享且高度保守的[Fe-S]组装机制的假设的证据。
Several biological activities depend on iron-sulfur clusters ([Fe-S]). Even though they are well-known in several organisms their function and metabolic pathway were poorly understood in the majority of the organisms. We propose to use the amoeba Dictyostelium discoideum, as a biological model to study the biosynthesis of [Fe-S] at the molecular, cellular and organism levels. First, we have explored the D. discoideum genome looking for genes corresponding to the subunits that constitute the molecular machinery for Fe-S cluster assembly and, based on the structure of the mammalian supercomplex and amino acid conservation profiles, we inferred the full functionality of the amoeba machinery. After that, we expressed the recombinant mature form of D. discoideum frataxin protein (DdFXN), the kinetic activator of this pathway. We characterized the protein and its conformational stability. DdFXN is monomeric and compact. The analysis of the secondary structure content, calculated using the far-UV CD spectra, was compatible with the data expected for the FXN fold, and near-UV CD spectra were compatible with the data corresponding to a folded protein. In addition, Tryptophan fluorescence indicated that the emission occurs from an apolar environment. However, the conformation of DdFXN is significantly less stable than that of the human FXN, (4.0 vs. 9.0 kcal mol-1, respectively). Based on a sequence analysis and structural models of DdFXN, we investigated key residues involved in the interaction of DdFXN with the supercomplex and the effect of point mutations on the energetics of the DdFXN tertiary structure. More than 10 residues involved in Friedreich\'s Ataxia are conserved between the human and DdFXN forms, and a good correlation between mutational effect on the energetics of both proteins were found, suggesting the existence of similar sequence/function/stability relationships. Finally, we integrated this information in an evolutionary context which highlights particular variation patterns between amoeba and humans that may reflect a functional importance of specific protein positions. Moreover, the complete pathway obtained forms a piece of evidence in favor of the hypothesis of a shared and highly conserved [Fe-S] assembly machinery between Human and D. discoideum.