Structure, Function and Dynamics of Heme Degrading Enzymes

描述(申请人提供)：铁是大多数生物体的基本营养物质，包括致病细菌。试图定居人类的病原体面临着极低浓度的游离铁。因此，许多病原体进化出了获取铁的复杂机制，包括利用血红素铁。此外，最近有研究表明，在感染的早期阶段，金黄色葡萄球菌更喜欢铁而不是血红素。因此，靶向病原菌从宿主吸收铁和血红素铁的途径是开发新抗生素的可行方法。在条件致病菌铜绿假单胞菌中，参与血红素吸收和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、动力学和蛋白质间的相互作用在很大程度上是未知的。在这一应用中，我们建议通过研究可溶性蛋白质的结构、功能、动力学和相互作用来填补这一空白，这些蛋白质有助于从血红蛋白中捕获血红素，并帮助降解铜绿假单胞菌胞浆中的血红素。这些研究的重要成果包括：(1)两个未知的电子传递蛋白(BFD和FPR)的生化和结构特征，我们推测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胞浆中释放其铁。我们还计划研究促进从BFD到血红素加氧酶的电子转移的蛋白质-蛋白质相互作用，以支持血红素的降解。(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，并将其运送到外膜受体进行内化。获得的HasAp的结构信息将被用来确定其与血红蛋白的相互作用(3)多肽动力学如何有助于铜绿假单胞菌血红素加氧酶的血红素氧化活性的表征，在这一点上，这在很大程度上是由于即将到期的资金周期中支持的工作而被更好地理解。从这些研究中获得的信息有望为未来设计新的治疗策略提供几个切入点，旨在干扰铜绿假单胞菌对血红素的吸收和降解。

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.