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。试图在人类中定居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，最近已经表明，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，有可能靶向病原菌从其宿主中同化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，许多参与血红素摄取和血红素利用的蛋白质具有指定的功能。然而，结构，动力学和蛋白质间的相互作用，促进宿主血红素捕获，内化和降解的胞质溶胶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、动力学和缔合来填补这一空白，所述可溶性蛋白质有助于从血红蛋白中捕获血红素并有助于在铜绿假单胞菌的胞质溶胶中降解血红素。本研究的主要结果如下：（1）对铜绿假单胞菌中两种未知的电子传递蛋白（Bfd和Fpr）进行了生物化学和结构表征，我们推测这两种蛋白的功能是提供血红素加氧酶所需的7个电子，从而在铜绿假单胞菌的胞质溶胶中切割血红素并释放铁。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的结构表征，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。所获得的HasAp的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有助于来自铜绿假单胞菌的血红素加氧酶的血红素氧化活性，在这一点上，由于在即将到期的资助周期中支持的工作，这一点得到了更好的理解。从这些调查中了解到的信息预计将提供几个切入点，为未来设计的新的治疗策略，旨在干扰血红素的吸收和降解铜绿假单胞菌。

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.