Structure, Function and Dynamics of Heme Degrading Enzymes

血红素降解酶的结构、功能和动力学

• 批准号: 7199450
• 负责人: Mario Rivera
• 金额: $ 26万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-04-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7199450
• 关键词: NAD(P)H oxidoreductase; Pseudomonas aeruginosa; X ray crystallography; active sites; biotransformation; chemical binding; cytochrome P450; cytochrome b5 reductase; divalent metal; electrochemistry; electron spin resonance spectroscopy; enzyme activity; enzyme structure; ferriheme; heme; heme oxygenase; hydrogen bond; intermolecular interaction; mitochondrial membrane; molecular shape; nuclear magnetic resonance spectroscopy; oxygen transport; protein structure function; site directed mutagenesis; stable isotope

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.

描述（由申请人提供）：铁是大多数生物（包括致病细菌）的必不可少的营养素。试图定居人类的致病细菌面临着极低浓度的游离铁。因此，许多病原体已经进化了采集铁的复杂机制，包括血红素铁的利用。此外，最近已经表明，在感染的早期，金黄色葡萄球菌更喜欢来自血红素的铁。因此，病原细菌用来从其宿主中吸收铁和血红素铁的靶向路径是一种可行的新抗生素开发方法。在机会性病原体铜绿假单胞菌中，许多参与血红素摄取和血红素利用的蛋白质已指定功能。但是，促进宿主 - 血红素捕获，内在化和降解的结构，动力学和蛋白质相互作用在很大程度上是未知的。在此应用中，我们建议通过研究可溶性蛋白的结构，功能，动力学和关联来填补这一空白，从而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细胞质中降解它。拟议的研究的重要结果是：（1）两个先前未知的电子转运蛋白（BFD和FPR）的生化和结构表征，我们假设功能可以传递血红素氧化酶所需的7个电子，以裂解血红素并释放其在铜绿假单胞菌中的铁并释放其铁。我们还计划研究促进电子从BFD转移到血红素氧酶的蛋白质蛋白相互作用，以支持血红素的降解。 （2）HASAP的结构表征，一种分泌的血红素结合蛋白，能够从血红蛋白中捕获血红素并将其传递到外膜受体中以进行内在化。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铜绿假单胞菌的血红素氧化酶的血红素氧化活性，此时，这在很大程度上可以理解，这在很大程度上是由于在到期资金周期中支持的工作。从这些研究中学到的信息预计将为未来的新型治疗策略设计提供多个入口点，以干扰铜绿假单胞菌的血红素摄取和降解。