Protein-Protein Interactions Involved in Siderophore Biosynthesis and Secretion

大多数维持生命的代谢过程都需要蛋白质之间的相互作用。此外，参与这些过程的代谢酶通常聚集成称为代谢物的大型复合物。将酶组织成代谢物可以提高代谢过程的效率，并有助于膜定位。我的实验室研究蛋白质-蛋白质相互作用在铁载体介导的细菌铁摄取中的作用。铁载体是细菌合成和分泌的小分子铁螯合剂，用于从细胞外环境中获取稀缺的铁。目前对参与铁载体生物合成的酶是如何在细菌细胞内组织的知之甚少。我们现在有证据表明，参与大肠杆菌铁载体肠杆菌蛋白生物合成的酶是在大肠杆菌细胞中作为代谢产物组织起来的。我们提出参与肠obactin生物合成和分泌的蛋白质之间的相互作用(i)通过底物通道增强代谢通量；（ii）通过代谢物和外排转运体之间的直接相互作用，将肠obactin代谢物锚定在大肠杆菌内膜上，促进分泌。我们将在成功的基础上，利用生物物理和结构方法，充分表征肠杆菌素生物合成（Ent）酶之间相互作用的性质。我们还将研究这种蛋白质相互作用如何通过底物通道增强代谢通量。从这些实验中获得的知识将使我们能够确定破坏Ent蛋白相互作用和底物通道过程的分子靶标。我们将研究这种破坏对细菌生长和铁载体分泌的影响。一些大肠杆菌菌株在肠obactin分泌前糖基化，以逃避宿主免疫系统。我们将研究肠obactin糖基转移酶IroB，以了解其功能如何与Ent生物合成机制协调。除了我们的生物物理和结构方法外，我们还将研究Ent生物合成机制如何在大肠杆菌细胞内定位。体内交联方法将用于鉴定与外排转运体接触的蛋白伙伴，并鉴定高阶Ent蛋白复合物。与显微镜专家合作，我们还将使用超分辨率显微镜和FRET显微镜来确定完整大肠杆菌细胞中Ent蛋白的共定位。本申请中提出的研究将确定破坏或增强大肠杆菌细胞中铁载体介导的铁摄取的目标。Ent-Ent和Ent-IroB蛋白相互作用的小分子干扰物可能导致保护家禽和牲畜的新型抗生素。这些过程的增强可能导致新的细菌菌株的工程可持续生物矿化铁和其他金属。

Most metabolic processes that support life require protein-protein interactions. Furthermore, metabolic enzymes that participate in such processes are often clustered into large complexes known as metabolons. Organization of enzymes into metabolons can enhance the efficiency of metabolic processes and can facilitate membrane localization. My laboratory studies the role of protein-protein interactions in siderophore-mediated bacterial iron uptake. Siderophores are small-molecule iron chelators that bacteria synthesize and secrete to obtain scarce iron from extracellular environment. Little is currently known about how the enzymes involved in siderophore biosynthesis are organized within a bacterial cell. We now have evidence that the enzymes involved in the biosynthesis of the E. coli siderophore enterobactin are organized as a metabolon in E. coli cells. We propose that interactions between proteins involved in enterobactin biosynthesis and secretion (i) enhance metabolic flux via substrate channeling and (ii) anchor the enterobactin metabolon to the E. coli inner membrane to facilitate secretion via direct interaction between the metabolon and an efflux transporter. We will build on our successful efforts to fully characterize the nature of interactions between enterobactin biosynthetic (Ent) enzymes using biophysical and structural approaches. We will also investigate how such protein interactions may enhance metabolic flux via substrate channeling. Knowledge gained from these experiments will allow us to identify molecular targets to disrupt Ent protein interactions and substrate channeling processes. We will study the effects of such disruptions on bacterial growth and siderophore secretion. Some E. coli strains glycosylate enterobactin prior to its secretion in order to evade host immune systems. We will study IroB, the enterobactin glycosyltransferase, to understand how its functions may be coordinated with the Ent biosynthetic machinery. In addition to our biophysical and structural approaches, we will investigate how the Ent biosynthetic machinery is localized within E. coli cells. An in vivo crosslinking approach will be used to identify protein partners in contact with the efflux transporter EntS, and to identify higher-order Ent protein complexes. In collaboration with microscopy experts, we will also use super-resolution microscopy and FRET microscopy to determine co-localization of Ent proteins in intact E. coli cells. The research proposed in this application will identify targets for either disruption or enhancement of siderophore-mediated iron uptake in E. coli cells. Small-molecule disruptors of Ent-Ent and Ent-IroB protein interactions may lead to novel antibiotics to protect poultry and livestock. Enhancement of these processes could lead to the engineering of novel bacterial strains for sustainable biomining of iron and other metals.