Structure and Function of Cyclodipeptide Oxidase-Like Enzyme Filaments

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.