CAREER: Establishing the Roles of Multi-enzyme Complexes in Metabolic Network Regulation

职业：建立多酶复合物在代谢网络调节中的作用

• 批准号: 1845451
• 负责人: Toshihiro Obata
• 金额: $ 74.7万
• 依托单位: University of Nebraska-Lincoln
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-01-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1845451&HistoricalAwards=false
• 关键词: CAREER; Establishing; Roles; Multi; enzyme

Enzymes that catalyze sequential reactions in a metabolic pathway often form a multi-enzyme complex. The complex allows intermediate metabolites to be channeled between enzymes. Theoretical and test tube-based experimental studies have shown that multi-enzyme complex formation is important in the regulation of metabolic networks. However, despite 40 years of research, the function of multi-enzyme complexes within a living cell are controversial and difficult to demonstrate due to difficulties controlling highly variable subcellular micro-environments, where these complexes reside. The goal of this project is to address this controversy by characterizing the function and role of multi-enzyme complexes as regulators of metabolic processes in living cells. The knowledge gained will help scientists understand how organisms maintain their metabolic homeostasis under fluctuating environments, and facilitate metabolic engineering of key pathways for synthetic biology applications. As part of the broader impacts of the project, the investigator will develop a creative paper craft hands-on activity to teach principles of enzyme-based metabolic regulation to a wide audience, including undergraduate and high school students, and adult audiences. This novel broader impact activity will positively impact literacy in metabolism and increase public engagement with metabolic research. In addition, the investigator will train graduate and undergraduate students through the project's research and educational activities. Rapid, flexible and coordinated regulation of metabolism is essential for all living organisms to respond to their changing environments which fluctuate sometimes within an order of seconds. Currently known molecular mechanisms cannot fully explain this regulatory process. In this project, the functionality of the malate dehydrogenase/ citrate synthase/aconitase multi-enzyme complex of the yeast Krebs tricarboxylic acid (TCA) cycle will be investigated as a model system. The overarching goal of this project is to experimentally establish the roles of the TCA cycle multi-enzyme complex in metabolic regulation in yeast cells. The hypothesis the investigator will explore is that the TCA cycle multi-enzyme complex enhances and/or redirects metabolic flux relative to the ratio of the associated enzyme complex. The bi-directional relationship between the rate of enzyme association and metabolic flux dynamics within the TCA cycle will be assessed through two research objectives: 1) assessment of the effects of variations in multi-enzyme complex affinity on metabolic flux, and 2) assessment of the effects of changes in metabolic flux dynamics on multi-enzyme complex formation. Metabolic flux in the TCA cycle and adjacent pathways will be analyzed using isotope tracer experiments and quantitative modeling methods. Enzyme complex association will be quantified using multiple techniques, including a novel bioluminescence-based real-time assay, which will be developed as part of this project. Results of this work will enhance the fundamental understanding of how enzyme kinetics couples with flux dynamics in a biochemical system, and further clarify the impact of subcellular micro-environments on systems level outcomes.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

在代谢途径中催化顺序反应的酶通常形成多酶复合体。该复合体允许中间代谢物在酶之间输送。理论和基于试管的实验研究表明，多酶复合体的形成在代谢网络的调节中起着重要作用。然而，尽管经过了40年的研究，但由于难以控制这些复合体所在的高度可变的亚细胞微环境，活细胞内多酶复合体的功能仍存在争议和难以证明。这个项目的目标是通过表征多酶复合体作为活细胞代谢过程调节器的功能和作用来解决这一争议。所获得的知识将帮助科学家了解生物体如何在波动的环境下保持其代谢动态平衡，并促进合成生物学应用的关键途径的代谢工程。作为该项目更广泛影响的一部分，研究人员将开发一个创造性的纸质手工动手活动，向包括本科生和高中生以及成年观众在内的广泛受众传授基于酶的代谢调节原理。这一新的更广泛的影响活动将对新陈代谢方面的扫盲产生积极影响，并增加公众对新陈代谢研究的参与。此外，调查员还将通过该项目的研究和教育活动培训研究生和本科生。对新陈代谢的快速、灵活和协调的调节对于所有生物体对其不断变化的环境做出反应是至关重要的，这些环境有时在几秒钟内波动。目前已知的分子机制不能完全解释这一调控过程。在本项目中，将以酵母Krebs三羧酸(TCA)循环中的苹果酸脱氢酶/柠檬酸合成酶/乌头酸酶多酶复合体为模型系统进行研究。该项目的首要目标是通过实验确定TCA循环多酶复合体在酵母细胞代谢调节中的作用。研究人员将探索的假设是，TCA循环多酶复合体相对于相关酶复合体的比例增强和/或重定向代谢流量。TCA循环中的酶结合速率和代谢流动力学之间的双向关系将通过两个研究目标来评估：1)评估多酶复合体亲和力变化对代谢流的影响；2)评估代谢流动力学变化对多酶复合体形成的影响。将使用同位素示踪实验和定量建模方法分析TCA循环和相邻途径中的代谢通量。将使用多种技术来量化酶复合体的结合，包括一种新的基于生物发光的实时分析，该方法将作为该项目的一部分进行开发。这项工作的结果将加强对生化系统中酶动力学与通量动力学如何耦合的基本理解，并进一步阐明亚细胞微环境对系统水平结果的影响。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

项目成果

Metabolites Modulate Malate Dehydrogenase‐Citrate Synthase Multienzyme Complex Formation

代谢物调节苹果酸脱氢酶-柠檬酸合酶多酶复合物的形成

• DOI: 10.1096/fasebj.2021.35.s1.01547
• 发表时间: 2021
• 期刊: The FASEB Journal
• 作者: omini, Joy;Obata, Toshihiro
• 通讯作者: Obata, Toshihiro

Malate Dehydrogenase‐Citrate Synthase Multienzyme Complex Dynamics Is Affected By TCA Cycle Flux In Living Yeast Cells

苹果酸脱氢酶 - 柠檬酸合酶多酶复合物动力学受活酵母细胞中 TCA 循环通量的影响

• DOI: 10.1096/fasebj.2022.36.s1.0r339
• 发表时间: 2022
• 期刊: The FASEB Journal
• 作者: Omini, Joy J.;Krassovskaya, Inga;Obata, Toshihiro
• 通讯作者: Obata, Toshihiro