Structure, Function and Dynamics of Heme Degrading Enzymes

描述（由申请人提供）：铁是大多数生物体（包括病原菌）的必需营养素。试图在人类中定殖的病原菌面临着极低浓度的游离铁。因此，许多病原体已经进化出复杂的铁获取机制，包括血红素铁的利用。此外，最近的研究表明，在感染的早期阶段，金黄色葡萄球菌更喜欢血红素中的铁。因此，靶向病原菌从宿主中吸收铁和血红素铁的途径可能是开发新抗生素的可行方法。机会性病原体铜绿假单胞菌中参与血红素摄取和血红素利用的许多蛋白质都具有指定的功能。然而，促进宿主血红素在细胞质中捕获、内化和降解的结构、动力学和蛋白质间相互作用在很大程度上是未知的。在本申请中，我们建议通过研究可溶性蛋白质的结构、功能、动力学和关联来填补这一空白，这些蛋白质有助于从血红蛋白中捕获血红素并帮助其在铜绿假单胞菌的细胞质中降解。拟议研究的重要成果是：（1）两种以前未知的电子传递蛋白（Bfd 和 Fpr）的生化和结构特征，我们假设它们的功能是传递血红素加氧酶所需的 7 个电子，以裂解血红素并在铜绿假单胞菌的细胞质中释放铁。我们还计划研究促进电子从 Bfd 转移到血红素加氧酶以支持血红素降解的蛋白质-蛋白质相互作用。 (2) HasAp 的结构特征，HasAp 是一种分泌型血红素结合蛋白，能够从血红蛋白中捕获血红素并将其递送至外膜受体进行内化。获得的 HasAp 结构信息将用于定义其与血红蛋白的相互作用 (3) 表征多肽动力学如何促进铜绿假单胞菌血红素加氧酶的血红素氧化活性，目前这一点得到了更好的理解，主要是由于即将到期的资助周期中支持的工作。从这些研究中获得的信息预计将为未来设计旨在干扰铜绿假单胞菌血红素摄取和降解的新型治疗策略提供几个切入点。

DESCRIPTION (provided by applicant): Iron is an essential nutrient for most organisms, including pathogenic bacteria. Pathogenic bacteria attempting to colonize humans are confronted with extremely low concentrations of free iron. Consequently, many pathogens have evolved sophisticated mechanisms for iron acquisition, including the utilization of heme-iron. Moreover, it has been recently shown that during the early stages of infection Staphylococcus aureus prefers iron from heme. Thus, it is possible that targeting paths used by pathogenic bacteria to assimilate iron and heme-iron from their host is a viable approach to de development of new antibiotics. Many of the proteins involved in heme uptake and heme utilization in the opportunistic pathogen Pseudomonas aeruginosa have designated functions. However, the structure, dynamics and inter-protein interactions that facilitate host-heme capture, internalization and degradation in the cytosol are largely unknown. In this application we propose to contribute to fill this gap by studying the structure, function, dynamics and association of the soluble proteins that aid in the capture of heme from hemoglobin and help degrade it in the cytosol of P. aeruginosa. Important outcomes of the proposed studies are: (1) Biochemical and structural characterization of two previously unknown electron transport proteins (Bfd and Fpr), which we hypothesize function to deliver the 7 electrons needed by heme oxygenase to cleave the heme and release its iron in the cytosol of P. aeruginosa. We also plan to investigate the protein-protein interactions that facilitate electron transfer from Bfd to heme oxygenase to support the degradation of heme. (2) Structural characterization of HasAp, a secreted heme binding protein capable of capturing heme from hemoglobin and delivering it to the outer membrane receptor for internalization. The acquired structural information of HasAp will be used to define its interactions with hemoglobin (3) Characterization of how polypeptide dynamics contribute to the heme oxidation activity of heme oxygenase from P. aeruginosa, which at this point is better understood largely due to work supported in the expiring funding cycle. The information learned from these investigations is expected to provide several entry points for the future design of novel therapeutic strategies aimed at interfering with heme uptake and degradation in P. aeruginosa.