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

在化学系生命过程化学项目的支持下，佛罗里达大学的丽贝卡·布彻将研究一系列酶，这些酶对线虫(蛔虫)中信号分子的产生至关重要。寄生线虫给农业造成了数十亿美元的损失，感染了世界上大约三分之一的人口。线虫通过分泌信号分子或信息素相互交流，这些分子或信息素也被称为蛔虫苷，使蠕虫能够协调它们的发育，以及各种行为，包括吸引、聚集和回避。本研究项目将研究羧酸酯酶(CEST)的酶机制、底物和蛋白质结构。这些酶负责调节影响其生物活性的蛔虫苷的特定化学修饰。对CEST酶的研究将有助于剖析线虫产生的大量信息素的生物学作用，并最终可能导致控制寄生线虫的新策略。该项目还将通过开发以课程为基础的本科生研究体验(CURE)来增加STEM(科学、技术、工程和数学)专业本科生的留存率，为新生在本科生涯早期提供真实的研究体验。本研究项目将通过异源表达、纯化和鉴定其对候选底物的酶活性来表征关键的CEST酶的生化活性。同时，该项目将通过喂食稳定同位素标记的蛔虫苷来表征体内CEST酶的底物范围。具有代表性的CEST酶将被结晶，并使用X射线结晶学确定其结构，以便研究其底物偏好的分子基础。由于蛔虫苷信息素中的修饰对它们的活性至关重要，了解这些修饰是如何被CEST酶结合的将有助于确定线虫信息素的生物学作用。此外，对CEST酶的酶活性的表征可以在未来实现线虫信息素和衍生物库的化学酶合成，这些信息素和衍生物库反过来可以用于针对寄生线虫。这一奖项反映了NSF的法定使命，并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，被认为值得支持。

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.