Characterization of a Family of Enzymes Responsible for the Modular Architecture of Nematode Pheromones

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With support from the Chemistry of Life Processes Program in the Division of Chemistry, Rebecca Butcher from the University of Florida will investigate a family of enzymes that are essential for the production of signaling molecules in nematodes (roundworms). Parasitic nematodes cause billions of dollars in agricultural damage and infect approximately one-third of the world’s human population. Nematodes communicate with each other by secreting signaling molecules or pheromones also known as ascarosides, that allow worms to coordinate their development, as well as various behaviors, including attraction, aggregation, and avoidance. This research project will study the enzymatic mechanisms, substrates, and protein structures of carboxylesterase (CEST) enzymes. These enzymes are responsible for mediating specific chemical modifications of ascarosides that influence their biological activities. Investigation of the CEST enzymes will be instrumental in dissecting the biological roles of the multitude of pheromones produced by nematodes and may ultimately lead to novel strategies for controlling parasitic nematodes. This project will also increase the retention of undergraduates in STEM (science, technology, engineering and mathematics) majors by developing a Course-based Undergraduate Research Experience (CURE) to provide freshmen with an authentic research experience early in their undergraduate careers. This research project will characterize the biochemical activities of key CEST enzymes by heterologously expressing them, purifying them, and characterizing their enzymatic activities against candidate substrates. In parallel, the project will characterize the substrate range of the CEST enzymes in vivo by feeding worms stable isotope-labeled ascarosides. Representative CEST enzymes will be crystallized, and their structures determined using X-ray crystallography in order to investigate the molecular basis for their substrate preferences. Because the modifications present in ascaroside pheromones are critical to their activities, knowledge of how these modifications are attached by CEST enzymes will help to determine the biological roles of nematode pheromones. Furthermore, characterization of the enzymatic activities of the CEST enzymes could in the future enable chemoenzymatic synthesis of nematode pheromones and libraries of derivatives, that could in turn be used to target parasitic nematodes.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.