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，东卡罗来纳大学的Holly Ellis教授和她的研究团队将研究一些酶进行的有趣的化学作用，这些酶参与获得细菌生存所需的元素硫。这些酶的一个令人着迷的特征是它们能够改变自己的结构，以促进它们催化的反应。许多这些关键的结构变化以及伴随而来的催化事件都鲜为人知。本研究选用的双组分酶系利用不同的结构变化促进硫的获取。因此，这些酶系统将是评估多种不同的结构策略的理想选择，这些策略将是催化所必需的。该项目还将与Brody Rise大学预科计划相结合，以培训科学中代表性较低群体的高中生了解酶功能所使用的不同生化技术。本科生奖学金将在夏季提供，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，依赖于黄素单核苷酸(FMN)的双组分单加氧酶对于细菌中硫的获取是必不可少的。这些酶利用FMN还原酶和单加氧酶来执行它们的关键功能角色；这些角色需要优雅的协调机制策略来进行脱磺。上述目标将评估硫获取过程中涉及的两组分黄素依赖系统是否进化出共同的结构特征和机械步骤，以发挥其复杂的功能作用。目标1是确定促进脱磺的双组分依赖于FMN的单加氧酶的机制策略。目的2是确定两组分NAD(P)H：FMN还原酶的结构性质如何决定功能。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磺机理提供证据，并确定有助于NAD(P)H：FMN还原中黄素转移的低聚变化。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.