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

对微生物途径中鲜为人知的蛋白质进行表征，对于增加我们对生物过程的了解，发现新的蛋白质功能和识别新的蛋白质靶点是非常重要的，这对于开发急需的新的抗菌分子是非常必要的。我的研究计划的长期目标是破译关键细菌过程中蛋白质和酶的结构-功能关系，例如铁和血红素的获取和代谢过程，以增加知识，并为抗击细菌感染提供新的靶点和方法。 我们的目标是描述一个8基因簇，该基因簇参与了从引起人类食源性感染的肠杆菌大肠杆菌O157：H7获得和利用血红素的过程。在过去的几年里，我们鉴定了3种胞质Chu蛋白(Chus、Chux和Chuy)，由于它们的个体功能尚未确定，并且对Chu途径的生物学了解很少，因此我们建议进行进一步的研究。我们的工作表明，Chus是一种利用分子氧和过氧化氢作为氧化剂来降解血红素的酶，并产生一种马鞭铁中间体，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，即卟啉的打开，是由一种新的机制催化的，该机制产生了三吡咯的卟啉产物、血酸、一氧化碳和游离铁。将使用动力学和光谱方法来研究从马鞭铁血红素中打开卟啉的新机制。为了验证在试管中检测到的这种反应也在体内发生，我们的目标是使用靶向代谢组学方法从本地细菌中鉴定卟啉降解产物。楚蛋白的功能(S)将在一个非感染性的生理模型中进行研究。最后，我们建议探索在细胞质Chu蛋白中预测的体外和体内蛋白质-蛋白质复合体，以了解它们对催化的重要性。 这项拟议的研究将增加我们对肠道细菌和其他拥有这一途径的细菌中涉及获取血红素-铁的关键酶的基础知识。我们建议的一个主要优点是，将在试管和细菌细胞中对Chu蛋白的活性和机制进行研究。因此，我们的结果将为评估这些蛋白质中的一个或多个作为潜在的新靶点(S)来开发抗菌策略提供坚实、可靠的基础。从长远来看，我们的研究可能会对新抗菌剂的开发产生重大影响，因此对人类健康和农业(动物健康)等经济部门都很重要。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，这些领域是学术界和生物技术行业的高需求领域。

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.