Mechanistic and Structural Studies of Flavin Dependent N-hydroxylating Monooxygenases

铁是大多数生物必需的营养物质；然而，由于铁在水中的溶解度很低，它以低浓度存在。一些微生物已经开发出一种复杂的系统，通过产生被称为铁载体的铁螯合分子来从环境中获取铁。N-羟化单加氧酶是一类尚未鉴定的黄素依赖性单加氧酶，参与赖氨酸和鸟氨酸侧链上氮原子的羟化反应，导致铁载体的生物合成。该项目将利用诱变和动力学研究、质谱分析和X射线结晶学来确定N-羟化单加氧酶家族的两个成员：N5-鸟氨酸和N6-赖氨酸羟基酶的结构和机制。最终目的是了解氨基酸羟化和选择性的机制以及蛋白质运动在催化中的作用。这些信息将有助于我们理解黄素酶激活氧气的机制，以及大自然如何利用含氧黄素中间体来实现特定的化学结果。这一应用程序的研究将作为教学和培训高中、本科生和研究生的平台，使他们能够发展分子生物学、机械酶学、生物化学和蛋白质化学的技能和知识。少数族裔学生将通过弗吉尼亚理工大学文凭后研究和教育国际的指导活动和最大化学生多样性计划纳入这项研究。本科生和研究生将通过参加意大利帕维亚大学蛋白质结晶学小组的国际实习计划获得结构生物化学方面的培训。参观实验室的高中生将与弗吉尼亚理工大学植物研究和教育伙伴关系一起，通过确定黄素单加氧酶突变体的生理作用，接触到基于假设的研究。

Iron is an essential nutrient for most living organisms; however, it is present in low concentration due to its low solubility in water. Some microbes have developed an elaborate system to acquire iron from the environment by producing iron-chelating molecules called siderophores. N-hydroxylating monooxygenases are a group of uncharacterized flavin-dependent monooxygenases involved in the hydroxylation of nitrogen atoms on the side chains of lysine and ornithine in the pathway leading to the biosynthesis of siderophores. This project will employ mutagenesis and kinetic studies, mass spectral analyses, and x-ray crystallography to determine the structure and mechanism of two members of the N-hydroxylating monooxygenase family: N5-ornithine and N6-lysine hydroxylases. The ultimate goal is to understand the mechanisms of amino acid hydroxylation and selectivity and the role of protein motion in catalysis. This information will contribute to our understanding of the mechanism of oxygen activation by flavoenzymes and how nature harnesses oxygenated flavin intermediates for specific chemical outcomes. The research in this application will serve as a platform for teaching and training high school, undergraduate, and graduate students, enabling them to develop skills and knowledge in molecular biology, mechanistic enzymology, biochemistry, and protein chemistry. Minority students will be integrated into this research through the mentoring activities of the PI in Virginia Tech's Post-Baccalaureate Research and Education and the Initiative to Maximize Student Diversity Programs. Undergraduate and graduate students will gain training in structural biochemistry by participating in an international internship program in the protein crystallography group at the University of Pavia, Italy. High school students visiting the laboratory will be exposed to hypothesis-based research by determining the physiological role of flavin monooxygenase mutants of A. thaliana in conjunction with the Partnership for Research and Education in Plants at Virginia Tech.