Heme Oxygenase: Structure, Function and Pathogenesis

描述(申请人提供)：病原菌需要铁才能生存和引起感染。血红素占宿主体内可利用铁的90%。因此，了解血红素获得和铁释放的机制将为开发新的治疗靶点提供所需的知识。革兰氏阴性和革兰氏阳性病原菌都进化出受体介导的血红素摄取系统，通过该系统获得铁。这个过程中的一个关键步骤是通过血红素加氧酶(HO)的作用将铁从血红素大循环中释放出来。该提案的具体目的是：a)从结构上表征来自白喉弧菌(CD-HO)、脑膜炎奈瑟菌(Nm-HO)和铜绿假单胞菌(pa-HO)的可溶性细菌HO酶。CD-HO在结构上与哺乳动物HO蛋白同源，并将作为更大的膜结合蛋白的模型系统。Nm-HO和Pa-HO代表了一类新的独特的HO酶，在Pa-HO的情况下表现出改变的区域选择性。利用X射线结晶学和核磁共振方法，我们将能够获得重要的见解：蛋白质的构象和动力学在血红素这一独特的酶家族的反应中的作用；b)通过定点突变和光谱研究相结合的方法来阐明血红素的羟基化和区域选择性的机制，其目的是确定在决定HO反应中的区域选择性和关键中间体的关键结构和电子因素方面的关键结构和电子因素；c)阐明马鞭铁血红素和胆绿素的形成机理和生物物理性质。阐明细菌HO蛋白的作用机制对于理解血红素的利用在发病机制中的作用以及未来开发抑制剂作为潜在的治疗药物将是至关重要的。

DESCRIPTION (provided by applicant): Pathogenic bacteria require iron for their survival and ability to cause infection. Heme comprises 90% of the iron available within the host. Therefore, understanding the mechanism of heme acquisition and iron release will provide the knowledge required for the development of new therapeutic targets. Both gram-negative and gram-positive pathogenic bacteria have evolved receptor mediated heme uptake systems by which they acquire iron. A key step in the process is the release of iron from the heme macrocycle by the action of heme oxygenase (HO). The specific aims of the proposal are to a) structurally characterize the soluble bacterial HO enzymes from C. diphtheriae (cd-HO), N. meningitides (nm-HO) and P. aeruginosa (pa-HO). The cd-HO is structurally homologous to the mammalian HO proteins and will serve as a model system for the larger membrane bound proteins. The nm-HO and pa-HO represent a new unique class of HO enzymes that in the case of pa-HO show an altered regioselectivity. Using both X-ray crystallographic and NMR methodologies we will be able to obtain significant insight into the role of protein conformation and dynamics in heme reactivity in this unique family of enzymes; b) elucidate the mechanism of heme hydroxylation and regioselectivity by a combination of site-directed mutagenesis and spectroscopic studies designed to identify key structural and electronic factors in determining both regioselectivity and the formation of a key intermediate in the HO reaction; c) To elucidate the mechanistic formation and biophysical properties of verdoheme and biliverdin. The elucidation of the mechanism of action of the bacterial HO proteins will be crucial in understanding the role of heme utilization in pathogenesis, as well as in future development of inhibitors as potential therapeutic agents.