Novel Imine Formation by ATP Grasp Enzymes

With the support of the Chemistry of Life Processes Program in the Division of Chemistry, Dr. Yousong Ding will collaborate with Drs. Steven Bruner and Gustavo Seabra, all from the University of Florida, to study the mechanism for the formation of an imine bond (carbon connected to nitrogen by two bonds) by members of an enzyme superfamily known as ATP grasp enzymes.

Imines are essential to a number of life processes and they are formed during many types of enzyme-catalyzed reactions. However, little is known about how enzymes in the ATP grasp enzyme superfamily catalyze imine formation. The ATP grasp superfamily has about 550,000 members, some of which catalyze reactions for central biological pathways, e.g., making proteins, cell-walls, and fatty acids.

Using a suite of biochemical, structural and computational tools, the research team will characterize how two ATP grasp enzymes promote the formation of imines for the production of complex molecules found in nature and discover new compounds and processes driven by imine-forming ATP grasp enzymes. This project aims to deliver novel insights into how nature has devised novel functions in the same enzyme family and reveal how these enzymes could aid in making complex molecules for applications in biotechnology.

Furthermore, this project will provide opportunities for training students, particularly women and underrepresented minorities, in modern scientific techniques, thereby preparing them for advanced careers in science. Through established collaborations with the Center for Undergraduate Research, Summer Undergraduate Research at Florida, and the Center for Precollegiate Education and Training at the University of Florida, this project will also engage high school and college students in the research and magnify its broader impacts through outreach activities for high school teachers.

The imine is important as a key reactive intermediate species in a number of enzyme catalyzed reactions and as an emerging functional group of bioactive small molecules. ATP grasp enzymes are known to form the amide, ester or thioester linkage. However, two ATP grasp enzymes MysC and MysD catalyze imine formation for the biosynthesis of natural UV protectants mycosporine-like amino acids (MAAs), representing new chemistry for this enzyme superfamily.

This project will investigate the molecular basis for fundamental deviations from the canonical reaction mechanism for ATP-dependent grasp enzymes that favor imine formation here. Specifically, the research team will characterize the reaction paths, substrate requirements, kinetic details, and unique structural features of MysC (Objective 1) and MysD (Objective 2), and explore new reaction manifolds and processes enabled by the new chemistry of ATP grasp enzymes (Objective 3).

As such, this work will likely add to our fundamental understanding of the consequences of protein sequence variation upon protein structure and function. Furthermore, the outlined studies will likely provide fresh information about the functional scope of the ATP grasp enzyme superfamily in nature, uncover hidden chemical diversity and biological processes, and open up longer term applications of these imine-forming enzymes in biotechnology.

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.