Structure and Function of Cyclodipeptide Oxidase-Like Enzyme Filaments

在化学系生命过程化学（CLP）项目的支持下，Tobias W。密歇根大学安阿伯分校的吉森正在研究在细菌和古细菌中发现的肽修饰酶丝的结构和功能。由非细胞骨架酶形成的纤维丝代表了控制细胞内代谢酶的活性或定位的进化保守机制。丝状蛋白酶直到最近才开始被详细描述，主要是由于其生物化学和生物物理分析的历史困难。拟议的工作将产生新的见解的结构和分子机制，管理的自组装和催化活性的酶丝。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饰酶丝奠定基础。该提案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）以及蛋白质丝的生物化学和生物物理分析方面的专业培训。该项目还将支持密歇根大学合成生物学本科团队。现代酶学和生物催化剂研究需要建立更好和更普遍的表达，纯化，表征和更充分开发丝状蛋白质的方法。该研究项目旨在探索不同的环二肽氧化酶（CDO）样酶形成细丝的能力，确定其高分辨率结构，并研究CDO样酶催化和底物范围。不同的蛋白质生物化学和生物物理技术将被用来研究细丝自组装。将进行单粒子冷冻电镜以确定CDO样酶丝的高分辨率结构。底物筛选环二肽，氨基酸，和相关的小分子将被用来探索底物范围和催化性质的CDO样酶丝。从这项研究的信息可以提供新的分子水平的深入了解CDO样酶丝形成的结构决定因素，底物选择性，和catalyst.This奖反映了NSF的法定使命，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。

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.