Coordinated Mechanistic Approaches to Desulfonation in Two-component FMN Monooxygenases

在化学系生命过程化学项目的支持下，东卡罗莱纳大学的Holly Ellis教授和她的研究小组将研究一些酶所进行的有趣的化学反应，这些酶参与了细菌生存所需元素硫的获取。这些酶的一个令人着迷的特点是它们能够改变它们的结构以促进它们催化的反应。许多这些关键的结构变化和伴随的催化事件知之甚少。本研究选择的双组分酶系统利用各种结构变化来促进硫的获取。因此，这些酶系统将是理想的，以评估多种不同的结构策略，这将是催化必不可少的。该项目还将与Brody RISE大学预科项目相结合，培训来自代表性不足群体的高中生，使用不同的生化技术来了解酶的功能。夏季将提供本科生奖学金，以指导和鼓励代表性不足的学生参与科学研究。当首选硫源有限时，双组分黄素单核苷酸（FMN）依赖的单加氧酶是细菌获得硫所必需的。这些酶利用FMN还原酶和单加氧酶来发挥其关键的功能作用；需要一个优雅的协调机制策略来脱硫的角色。所描述的目标将评估是否参与硫获取的双组分黄素依赖系统已经进化出共同的结构特征和机械步骤来执行其复杂的功能角色。目的1是确定促进脱硫的双组分fmn依赖单加氧酶的机制策略。目的2是确定双组分NAD(P)H:FMN还原酶的结构性质如何决定其功能。这两个目标都利用互补的光谱、动力学和生物物理方法来为脱硫机制提供证据，并确定有助于NAD(P)H:FMN还原酶中黄素转移的低聚物变化。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识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, Professor Holly Ellis and her research team at East Carolina University will study the interesting chemistry carried out by some of the enzymes involved in the acquisition of the element sulfur that bacteria need for survival. A fascinating feature of these enzymes is their ability to change their structures to facilitate the reactions they catalyze. Many of these crucial structural changes and accompanying catalytic events are poorly understood. The two-component enzyme systems selected for this study utilize various structural changes to promote sulfur acquisition. Therefore, these enzyme systems will be ideal to evaluate a multitude of different structural strategies that would be essential for catalysis. The project will also be integrated with the Brody RISE Pre-College Program to train high school students from underrepresented groups in the sciences in different biochemical techniques used to understand enzyme function. An undergraduate student fellowship will be provided in the summer to mentor and encourage participation of underrepresented students in scientific research. Two-component flavin mononucleotide(FMN)-dependent monooxygenases are essential for sulfur acquisition in bacteria when preferred sulfur sources are limiting. These enzymes utilize an FMN reductase and monooxygenase to carry out their critical functional roles; roles that require an elegant coordinated mechanistic strategy for desulfonation. The described objectives will evaluate if the two-component flavin-dependent systems involved in sulfur acquisition have evolved common structural features and mechanistic steps to carry out their complex functional role. Objective 1 is to identify the mechanistic strategies of two-component FMN-dependent monooxygenases that promote desulfonation. Objective 2 is to determine how the structural properties of two-component NAD(P)H:FMN reductases dictate function. Both objectives utilize complementary spectroscopic, kinetic, and biophysical approaches to provide evidence for the desulfonation mechanism and identify oligomeric changes that contribute to flavin transfer in the NAD(P)H:FMN reductases.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.