Structure and Function of Cyclodipeptide Oxidase-Like Enzyme Filaments

密歇根大学安娜堡分校的Tobias W. Giessen在化学系生命过程化学（CLP）项目的支持下，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是一种控制细胞内代谢酶活性或定位的进化保守机制。丝状酶直到最近才开始被详细地描述，这主要是由于它们在生化和生物物理分析方面的历史困难。所提出的工作将对控制酶丝自组装和催化活性的结构和分子机制产生新的见解。这些新发现可以为肽修饰酶丝在可持续生物催化和化学酶合成中的应用奠定基础。该计划将使本科生和博士后获得冷冻电子显微镜（cryo-EM）和蛋白质细丝的生化和生物物理分析的专业培训。该项目也将支持本科密歇根合成生物学团队。在现代酶学和生物催化剂研究中，需要建立更好、更通用的表达、纯化、表征和更充分开发丝状蛋白的方法。本研究项目旨在探索多种环二肽氧化酶（CDO）样酶形成细丝的能力，确定其高分辨率结构，并研究CDO样酶的催化作用和底物范围。多种蛋白质生化和生物物理技术将用于研究纤维自组装。单颗粒低温电镜将进行确定高分辨率结构的cdo样酶丝。环二肽、氨基酸和相关小分子的底物筛选将用于探索cdo样酶丝的底物范围和催化性能。这项研究的信息可以提供新的分子水平的洞察结构决定因素的cdo样酶丝的形成，底物选择性和催化。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.