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

细菌中血红素铁获取和利用的蛋白质和酶的生化和功能表征

• 批准号: RGPIN-2019-05182
• 负责人: Couture, Manon
• 金额: $ 2.33万
• 依托单位: Université Laval
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=744409
• 关键词: Biochemical; functional; characterization; proteins; enzymes

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.

对微生物中尚不清楚的途径的蛋白质的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鉴定新的蛋白质靶标是重要的，这对于帮助开发急需的新的抗微生物分子是非常期望的。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标和方法来对抗细菌感染。 我们的目标是表征一个8基因簇参与血红素收购和利用肠杆菌大肠杆菌O 157：H7，导致食源性感染的人类。在过去的几年中，我们表征了3种细胞质Chu蛋白（ChuS，ChuX和ChuY），我们建议进一步研究，因为它们的单独功能尚未建立，并且Chu途径的生物学知之甚少。我们的工作表明，ChuS是一种酶，使用分子氧和过氧化氢作为氧化剂降解血红素，并产生verdoheme中间体，这是由众所周知的血红素加氧酶产生的相同中间体。然而，下一步，卟啉的开放，是由一种新的机制，产生三吡咯卟啉产品，血红素酸，一氧化碳和游离铁催化。这种新的机制开放的卟啉从绿血红素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也发生在体内，我们的目标是使用有针对性的代谢组学方法来识别来自天然细菌的卟啉降解产物。将在非感染性生理模型中研究Chu蛋白的功能。最后，我们建议在体外和体内的蛋白质-蛋白质复合物的细胞质楚蛋白之间的预测，以了解它们的重要性，催化探测。 这项研究将增加我们对肠细菌和其他具有这种途径的细菌中血红素铁获得关键酶的基本知识。我们建议的一个主要优势是，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，我们的研究结果将提供一个坚实的，可靠的基础，以评估这些蛋白质的一个或多个潜在的新目标（S）的抗菌策略的发展。从长远来看，我们的研究可能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）等经济部门至关重要。这项研究还将有助于在蛋白质科学，生物化学和生理学的动态领域谁是在学术界和生物技术行业的高需求的高素质人才的培训。