Enzymes of Ornithine-Hydroxamate Siderophores

几乎所有的细菌都需要铁来生长。了解细菌获取铁的主要过程可以为了解如何破坏铁摄取过程提供有价值的见解，以及限制感染性微生物生长的潜在新策略。本提案的重点是铁载体的生物合成。铁载体是细菌产生的天然物质，它能将铁紧密结合，促进细菌对铁的吸收。该提案的目标是弄清楚细菌是如何从简单的构建块逐步产生这些化合物的。该研究计划旨在了解一种被称为pyoverdin的特定铁载体的三个步骤。本提案的部分内容旨在使本科生获得有意义的研究经验，以激发研究事业。该提案还包括一个由研究生进行的项目，为化学和生物学界面的未来科学家提供培训和指导。堪萨斯大学正在进行的项目将帮助积极招募未被充分代表的少数民族担任本科研究职位。PI还积极参与指导和招募堪萨斯大学研究生项目的不同学生，并参与社区外展活动，以激发即使是最年轻的（学龄前）学生对科学的兴趣。该奖项由生命过程化学项目化学部颁发给堪萨斯大学的奥黛丽·兰姆博士，为研究羟基酸铁分子生物合成的酶提供了资金。研究中的第一种酶，鸟氨酸羟化酶，是一种B类黄蛋白单加氧酶，具有有趣的性质，使其成为催化循环中黄素-氧中间体晶体可视化的绝佳系统。第二种要研究的酶是羟基鸟氨酸甲酰基转移酶，一种假定的10-甲酰基四氢叶酸依赖的甘氨酸酰胺核糖核苷酸转化酶样酶，我们将为这类酶提供第一个生化和结构表征。最后，在一个由本科生驱动的项目中，我们将研究非核糖体肽合成酶PvdJ的腺苷化和缩合结构域的底物特异性。这两个模块NRPS结合了研究中的前两个酶产生的甲酰基-羟基鸟氨酸，以及赖氨酸。实验方法包括稳态和瞬态动力学，以及x射线晶体学。该项目的结果将显著促进对催化所需的结构-功能关系的理解。此外，该提案旨在解决我们在黄素化学，n -甲酰化化学以及这三种酶中生物活性肽形成方面的知识空白。

Nearly all bacteria require iron to grow. Understanding the main processes by which bacteria gain iron provides valuable insight into how to disrupt the iron uptake process, and potential new strategies to limit growth of infectious microbes. The focus of this proposal is the biosynthesis of siderophores. Siderophores are natural materials produced by bacteria that bind iron very tightly, and facilitate iron uptake by the bacterium. The goal of the proposal is to figure out how bacteria make these compounds stepwise from simple building blocks. The research plan aims to understand three of these steps for a specific siderophore called pyoverdin. Portions of this proposal are structured such that undergraduate students will obtain meaningful research experiences to inspire careers in research. This proposal also includes a project to be conducted by a graduate student, providing training and mentoring of future scientists at the interface of chemistry and biology. Ongoing programs at the University of Kansas will aid in the active recruitment of under-represented minorities to undergraduate research positions. The PI is also an active participant in mentoring and recruiting students of diversity to graduate programs at the University of Kansas, and is involved in community outreach to inspire an interest in science in even the youngest (preschool) students.This award to Dr. Audrey Lamb of the University of Kansas by the Chemistry of Life Processes Program in the Chemistry Division provides funding to investigate the enzymes of hydroxamate siderophore biosynthesis. The first enzyme under study, the ornithine hydroxylase, is a class B flavoprotein monooxygenase with interesting properties that make it an excellent system for crystallographic visualization of the flavin-oxygen intermediates of the catalytic cycle. The second enzyme to be studied is the hydroxyornithine formyl-transferase, a putative 10-formyl-tetrahydrofolate-dependent glycinamide ribonucleotide transformylase-like enzyme, for which we will provide the first biochemical and structural characterizations for this enzyme class. Finally, in a project driven by undergraduate researchers, we will study the substrate specificity of the adenylation and condensation domains of the nonribosomal peptide synthetase PvdJ. This two module NRPS incorporates the formyl-hydroxyornithine generated by the first two enzymes under study, as well as a lysine. The experimental approaches include steady state and transient state kinetics, and x-ray crystallography. The outcomes of this project will significa ntly advance the understanding of the structure-function relationships required for catalysis. Furthermore, this proposal is designed to address our gaps in knowledge regarding flavin chemistry, N-formylation chemistry, and the formation of bioactive peptides in these three enzyme classes.