Novel Imine Formation by ATP Grasp Enzymes

在化学系生命过程化学项目的支持下，丁友松博士将与来自佛罗里达大学的Steven Bruner博士和Gustavo Seabra博士合作，研究一种被称为ATP抓酶的酶超家族成员形成亚胺键（碳通过两个键连接到氮）的机制。亚胺是许多生命过程中必不可少的，它们在许多类型的酶催化反应中形成。然而，很少有人知道如何在ATP抓酶超家族的酶催化亚胺的形成。ATP抓握超家族有大约550，000个成员，其中一些催化中心生物途径的反应，例如，制造蛋白质细胞壁和脂肪酸利用一套生物化学，结构和计算工具，研究小组将表征两种ATP抓酶如何促进亚胺的形成，以生产自然界中发现的复杂分子，并发现由亚胺形成ATP抓酶驱动的新化合物和过程。该项目旨在提供新的见解，了解自然界如何在同一个酶家族中设计新的功能，并揭示这些酶如何帮助制造复杂的分子，以应用于生物技术。此外，该项目将为学生，特别是妇女和代表人数不足的少数民族提供现代科学技术培训机会，从而为他们在科学领域的高级职业生涯做好准备。通过与本科生研究中心、佛罗里达夏季本科生研究中心和佛罗里达大学预科教育和培训中心的合作，该项目还将吸引高中和大学生参与研究，并通过对高中教师的推广活动扩大其更广泛的影响。亚胺是许多酶催化的关键反应中间体，反应和作为一个新兴的功能团的生物活性小分子。已知ATP抓取酶形成酰胺、酯或硫酯键。然而，两种ATP抓酶MysC和MysD催化亚胺形成，用于天然紫外线保护剂类菌孢菌素氨基酸（MAA）的生物合成，代表了这种酶超家族的新化学。本项目将研究从ATP依赖的抓酶的典型反应机制，有利于亚胺形成在这里的根本偏差的分子基础。 具体而言，研究小组将表征MysC（目标1）和MysD（目标2）的反应路径、底物要求、动力学细节和独特的结构特征，并探索由ATP抓取酶的新化学实现的新反应歧管和过程（目标3）。因此，这项工作可能会增加我们对蛋白质序列变异对蛋白质结构和功能的影响的基本理解。此外，概述的研究可能会提供有关ATP抓酶超家族在自然界中的功能范围的新信息，揭示隐藏的化学多样性和生物过程，并打开这些亚胺形成酶在生物技术中的长期应用。该奖项反映了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 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.