Enzymes of Ornithine-Hydroxamate Siderophores

鸟氨酸异羟肟酸铁载体酶

• 批准号: 1403293
• 负责人: Audrey Lamb
• 金额: $ 60.6万
• 依托单位: University of Kansas Center for Research Inc
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-15 至 2018-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1403293&HistoricalAwards=false
• 关键词: Enzymes; Ornithine; Hydroxamate; Siderophores

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.

几乎所有的细菌都需要铁来生长。了解细菌获得铁的主要过程为如何破坏铁吸收过程以及限制感染性微生物生长的潜在新策略提供了有价值的见解。 该提案的重点是铁载体的生物合成。 铁载体是由细菌产生的天然物质，其非常紧密地结合铁，并促进细菌对铁的吸收。 该提案的目标是弄清楚细菌如何从简单的构建模块逐步制造这些化合物。 该研究计划旨在了解一种名为绿脓菌荧光素的特定铁载体的三个步骤。 该提案的部分内容是这样构建的，即本科生将获得有意义的研究经验，以激发研究事业。 该提案还包括一个将由一名研究生开展的项目，为未来的科学家提供化学和生物学方面的培训和指导。 堪萨斯大学正在进行的项目将帮助积极招募代表性不足的少数民族担任本科研究职位。 PI还积极参与指导和招募多样性学生参加堪萨斯大学的研究生课程，并参与社区外展活动，以激发即使是最年轻的人对科学的兴趣，（学前班）这项由化学部生命过程计划化学授予堪萨斯大学的奥黛丽·兰姆博士的奖项为研究异羟肟酸铁载体的酶提供了资金生物合成 第一个酶的研究，鸟氨酸羟化酶，是一个有趣的性质，使其成为一个很好的系统，用于结晶可视化的催化循环的黄素-氧中间体的B类黄素蛋白单加氧酶。 第二个酶的研究是羟基鸟氨酸甲酰转移酶，一个假定的10-甲酰-四氢叶酸依赖性甘氨酰胺核糖核苷酸转化酶样酶，我们将提供第一个生化和结构表征这类酶。 最后，在一个由本科研究人员驱动的项目中，我们将研究非核糖体肽合成酶PvdJ的腺苷酸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种酶产生的甲酰基-羟基鸟氨酸，以及赖氨酸。 实验方法包括稳态和瞬态动力学，和X射线晶体学。 该项目的成果将显著推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-乙酰基化学以及这三类酶中生物活性肽形成方面的知识空白。