Structure and Function of Cyclodipeptide Oxidase-Like Enzyme Filaments

环二肽氧化酶样酶丝的结构和功能

• 批准号: 2400768
• 负责人: Tobias Giessen
• 金额: $ 59.79万
• 依托单位: Regents of the University of Michigan - Ann Arbor
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2024
• 资助国家: 美国
• 起止时间: 2024-05-01 至 2027-04-30
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2400768&HistoricalAwards=false
• 关键词: Structure; Function; Cyclodipeptide; Oxidase; Like

With the support of the Chemistry of Life Processes (CLP) program in the Division of Chemistry, Tobias W. Giessen of the University of Michigan, Ann Arbor is investigating the structure and function of peptide-modifying enzyme filaments found in both bacteria and archaea. Filament formation by non-cytoskeletal enzymes represents an evolutionarily conserved mechanism to control the activity or localization of metabolic enzymes inside cells. Filamentous enzymes have only recently begun to be characterized in detail, largely due to the historic difficulties in their biochemical and biophysical analysis. The proposed work will generate new insights into the structure and molecular mechanisms that govern the self-assembly and catalytic activity of enzyme filaments. These newly generated insights could lay the groundwork for using peptide-modifying enzyme filaments in sustainable biocatalysis and chemoenzymatic synthesis applications. This proposal will enable undergraduates and postdoctoral fellows to acquire specialized training in cryo-electron microscopy (cryo-EM) and the biochemical and biophysical analysis of protein filaments. This project will also support the undergraduate Michigan Synthetic Biology Team.There is a need in modern enzymology and biocatalysts research to establish better and more generalizable methods for expressing, purifying, characterizing and more fully developing filamentous proteins. This research project seeks to explore the ability of diverse cyclodipeptide oxidase (CDO)-like enzymes to form filaments, determine their high-resolution structures, and investigate CDO-like enzyme catalysis and substrate range. Diverse protein biochemical and biophysical techniques will be used to study filament self-assembly. Single particle cryo-EM will be carried out to determine high-resolution structures of CDO-like enzyme filaments. Substrate screens of cyclic dipeptides, amino acids, and related small molecules will be employed to explore the substrate range and catalytic properties of CDO-like enzyme filaments. Information from this study could provide new molecular level insight into the structural determinants of CDO-like enzyme filament formation, substrate selectivity, and catalysis.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.

在化学过程中的化学过程（CLP）方案的支持下，密歇根大学的Tobias W. Giessen Ann Arbor正在研究细菌和古细菌中发现的肽修饰酶丝的结构和功能。非细胞骨架酶形成细丝，代表了一种进化保守的机制，可以控制细胞内代谢酶的活性或定位。丝状酶直到最近才开始详细介绍，这主要是由于其生化和生物物理分析中的历史困难。提出的工作将产生有关控制酶丝的自组装和催化活性的结构和分子机制的新见解。这些新产生的见解可以为在可持续的生物催化和化学酶合成应用中使用肽修改酶的酶奠定基础。该建议将使本科生和博士后研究员能够获得低温 - 电子显微镜（Cryo-EM）的专业培训，以及对蛋白质丝的生化和生物物理分析。该项目还将支持密歇根州本科生合成生物学团队。在现代酶学和生物催化剂研究中，需要建立更好，更具概括性的方法来表达，纯化，表征和更全面的丝状蛋白质。该研究项目旨在探索类似酶（CDO）形成细丝，确定其高分辨率结构并研究类似CDO的酶催化和底物范围的各种环肽氧化酶（CDO）的能力。多种蛋白质生化和生物物理技术将用于研究丝自组装。将进行单个颗粒冷冻EM以确定类似CDO的酶丝的高分辨率结构。将使用环状二肽，氨基酸和相关小分子的底物筛选来探索CDO样酶丝的底物范围和催化特性。这项研究的信息可以提供有关CDO样酶丝形成，底物选择性和催化的结构决定因素的新分子水平。该奖项反映了NSF的法定任务，并被认为是值得通过基金会的知识分子优点和更广泛影响的审查标准来通过评估来获得支持的。