Biochemical and functional characterization of proteins and enzymes for heme-iron acquisition and utilisation in bacteria

对微生物中鲜为人知的途径的蛋白质进行表征，对于增加我们对生物过程的认识，发现新的蛋白质功能和鉴定新的蛋白质靶点非常重要，这对于帮助开发急需的新型抗菌分子是非常可取的。我的研究计划的长期目标是破译关键细菌过程的蛋白质和酶的结构-功能关系，例如铁和血红素的获取和代谢，以增加知识并提供新的靶点和方法来对抗细菌感染。******我们的目标是表征一个8个基因簇参与血红素获取和利用肠杆菌大肠杆菌O157:H7，导致人类食源性感染。在过去的几年里，我们鉴定了3种细胞质Chu蛋白（ChuS， ChuX和ChuY），我们建议进一步研究，因为它们的个体功能仍然不确定，并且对Chu通路的生物学知之甚少。我们的工作表明，ChuS是一种酶，它利用分子氧和过氧化氢作为氧化剂降解血红素，并产生一种血红素中间体，这与众所周知的血红素加氧酶产生的中间体相同。然而，下一步，卟啉的打开，是由一种新的机制催化生成三吡咯卟啉产物，血液酸，一氧化碳和游离铁。这种从毒芹素中打开卟啉的新机制将使用动力学和光谱方法进行研究。为了验证在试管中检测到的这种反应在体内也会发生，我们的目标是使用靶向代谢组学方法从天然细菌中鉴定卟啉降解产物。Chu蛋白的功能将在非感染性生理模型中进行研究。最后，我们建议在胞质Chu蛋白中进行体外和体内蛋白复合物的探测，以了解它们对催化的重要性。******提出的研究将增加我们对肠杆菌中血红素铁获取的关键酶的基础知识，以及其他具有这种途径的细菌。我们建议的一个主要优势是，对Chu蛋白的活性和机制的研究将在试管和细菌细胞中进行。因此，我们的研究结果将为评估这些蛋白质中的一种或多种作为开发抗菌策略的潜在新靶点提供坚实可靠的基础。从长远来看，我们的研究可能对开发新的抗菌剂产生重大影响，从而对人类健康和农业（动物健康）等经济部门具有重要意义。这项研究还将有助于培养蛋白质科学、生物化学和生理学等动态领域的高素质人才，这些人才在学术界和生物技术工业中需求量很大。*****

Characterization of proteins of poorly understood pathways from microorganisms is important to increase our knowledge of biological processes, to discover new protein functions and to identify new protein targets, which are highly desirable to help the development of much needed new antimicrobial molecules. The long-term objective of my research program is to decipher the structure-function relationships of proteins and enzymes of key bacterial processes, such as that of iron and heme acquisition and metabolism, in order to increase knowledge and to provide new targets and approaches to fight bacterial infections. ******We aim to characterize an 8-gene cluster involved in heme acquisition and utilisation from the enterobacterium Escherichia coli O157:H7 that causes food-borne infections in humans. We characterized in the past years 3 of the cytoplasmic Chu proteins (ChuS, ChuX and ChuY) and we propose further studies as their individual function is still not established and the biology of the Chu pathway is poorly understood. Our work shows that ChuS is an enzyme that degrades heme using molecular oxygen and hydrogen peroxide as the oxidants and producing a verdoheme intermediate, which is the same intermediate produced by the well-known heme oxygenase. However, the next step, the opening of the porphyrin, is catalyzed by a novel mechanism that generates a tripyrrole porphyrin product, hematinic acid, carbon monoxide and free iron. This novel mechanism of opening of the porphyrin from the verdoheme will be investigated using kinetic and spectroscopic approaches. To validate that this reaction detected in a test tube also occurs in vivo, we aim to identify the porphyrin degradation products from the native bacterium using a targeted metabolomics approach. The function(s) of the Chu proteins will be investigated in a non-infectious physiological model. Finally, we propose to probe in vitro and in vivo protein-protein complexes predicted among the cytoplasmic Chu proteins to understand their importance for catalysis. ******The proposed research will increase our fundamental knowledge of key enzymes involved in heme-iron acquisition in enterobacteria, and in other bacteria that possess such pathway. A major strength of our proposal is that the investigation of the activities and mechanisms of Chu proteins will be performed both in the test tube and in the bacterial cells. Hence, our results will provide a solid, reliable basis to evaluate one or more of these proteins as potential novel target(s) for the development of antimicrobial strategies. On the longer term, our studies may have a significant impact for the development of new antimicrobials, thus to be important to human health and to economic sectors such as agriculture (animal health). This research will also contribute to the training of highly qualified personnel in the dynamic areas of protein science, biochemistry and physiology who are in high demand in academia and biotechnology industries.*****