RUI: Nitrogenase and friends: Uncovering how diazotrophs regulate and maintain nitrogenase activity under unfavorable environmental conditions

RUI：固氮酶和朋友：揭示固氮菌如何在不利的环境条件下调节和维持固氮酶活性

• 批准号: 1905399
• 负责人: Cedric Owens
• 金额: $ 32.5万
• 依托单位: Chapman University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1905399&HistoricalAwards=false
• 关键词: RUI; Nitrogenase; friends; Uncovering; how

Nitrogenase is an enzyme found in certain bacteria, called diazotrophs, that converts nitrogen gas into ammonia, an essential nutrient for plant growth and, thus, nitrogenase is critical to agriculture. With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Cedric Owens of Chapman University to determine how diazotrophs maintain nitrogenase activity under environmental conditions that would typically be expected to inactivate the enzyme. Nitrogenase is inhibited by both carbon monoxide, a metabolic product, and oxygen, an essential gas for aerobic respiration in many diazotrophs. This project studies the countermeasures diazotrophs have taken to protect nitrogenase from inactivation by carbon monoxide, and how nitrogenase activity is regulated by cellular levels of oxygen and oxidizing compounds. Results from this project will enhance the understanding of nitrogenase activity in diazotrophs and potentially inform the design of synthetic nitrogen fixation systems that are more resistant to environmental inactivation. This project broadens participation of undergraduate students in research at Chapman University (a 4-year college) and at Santa Ana College (a 2-year minority serving college). Furthermore, a faculty member from Santa Ana College participates in research activities through a summer workshop and, together with the Dr. Owens, develops biochemistry experiments to teach fundamental chemical concepts to first-year general chemistry students. The two nitrogen fixing proteins being studied in this project are CowN, which responds to carbon monoxide (CO), and alpha-proteobacterial NifA, which responds to cellular oxygen and/or oxidation-reduction (redox) levels. While CO is a potent inhibitor of molybdenum containing nitrogenase (Mo-nitrogenase, the most common nitrogenase), it is a substrate that is reduced to short-chain hydrocarbons by the alternative vanadium-containing nitrogenase (V-nitrogenase). The mechanistic reason for the differing effects is unknown and is not explained by the enzymes' dissimilar metal compositions. Interestingly, Mo-nitrogenase tolerates CO in the presence of a small protein, CowN, which is expressed in a CO-dependent manner. The first aim of this project is to apply enzymological, biophysical and structural approaches to uncover the mechanism by which CowN protects Mo-nitrogenase from CO inhibition and determine if CowN alters Mo-nitrogenase reactivity. NifA is the main transcriptional regulator of nitrogenase, sensing either redox levels, oxygen levels, or both. The second aim is to use structural biology, spectroscopic and biophysical tools, in order to test the hypothesis that redox and/or oxygen levels are sensed through a previously uncharacterized iron-sulfur cluster via a non-canonical environmental sensing mechanism. The results of the project are expected to yield critical information on the cellular conditions required for nitrogenase expression.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.

氮酶是一种在重氮营养菌中发现的酶，它能将氮气转化为氨，氨是植物生长所必需的营养物质，因此，氮酶对农业至关重要。有了这个奖项，化学部门的生命过程化学项目资助了查普曼大学的Cedric Owens博士，以确定重氮养生物是如何在通常会使酶失活的环境条件下维持氮酶活性的。氮酶被一氧化碳（一种代谢产物）和氧气（许多重氮营养体中有氧呼吸所必需的气体）共同抑制。本项目研究重氮营养体保护氮酶免受一氧化碳失活的对策，以及氮酶活性如何受细胞氧和氧化性化合物水平的调节。该项目的结果将增强对重氮营养体中固氮酶活性的了解，并可能为设计更耐环境失活的合成固氮系统提供信息。这个项目扩大了本科生在查普曼大学（四年制学院）和圣安娜学院（两年制少数民族学院）的研究参与。此外，圣安娜学院的一名教员通过夏季研讨会参与研究活动，并与欧文斯博士一起开发生物化学实验，向一年级普通化学学生教授基本的化学概念。本项目研究的两种固氮蛋白是响应一氧化碳（CO）的CowN蛋白和响应细胞氧气和/或氧化还原（氧化还原）水平的α -蛋白质细菌NifA蛋白。虽然CO是含钼氮酶（mo -氮酶，最常见的氮酶）的有效抑制剂，但它是一种底物，可被替代的含钒氮酶（v -氮酶）还原为短链碳氢化合物。产生不同效果的机制原因尚不清楚，也不能用酶不同的金属成分来解释。有趣的是，mo -氮酶在一种以CO依赖方式表达的小蛋白CowN的存在下耐受CO。该项目的第一个目标是应用酶学，生物物理和结构方法来揭示CowN保护mo -氮酶免受CO抑制的机制，并确定CowN是否改变mo -氮酶的反应性。NifA是氮酶的主要转录调节因子，可感应氧化还原水平、氧水平或两者兼而有之。第二个目标是使用结构生物学、光谱学和生物物理学工具，以测试氧化还原和/或氧水平通过非规范环境感知机制通过以前未表征的铁硫簇来感知的假设。该项目的结果预计将产生对细胞条件所需的氮酶表达的关键信息。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。

项目成果

The structure of a Lactobacillus helveticus chlorogenic acid esterase and the dynamics of its insertion domain provide insights into substrate binding

• DOI: 10.1002/1873-3468.14731
• 发表时间: 2023-10-02
• 期刊: FEBS LETTERS
• 影响因子: 3.5
• 作者: Omori，Kellie K.;Drucker，Charles T.;Owens，Cedric P.
• 通讯作者: Owens，Cedric P.

Preventing chlorogenic acid quinone-induced greening in sunflower cookies by chlorogenic acid esterase and thiol-based dough conditioners

通过绿原酸酯酶和硫醇基面团调节剂防止向日葵饼干中绿原酸醌引起的变绿

• DOI: 10.1016/j.lwt.2022.114392
• 发表时间: 2023
• 期刊: LWT
• 作者: Pepra-Ameyaw, Nana Baah;Lo Verde, Christine;Drucker, Charles T.;Owens, Cedric P.;Senger, Lilian W.
• 通讯作者: Senger, Lilian W.

A highly active esterase from Lactobacillus helveticus hydrolyzes chlorogenic acid in sunflower meal to prevent chlorogenic acid induced greening in sunflower protein isolates

来自瑞士乳杆菌的高活性酯酶水解向日葵粕中的绿原酸，以防止向日葵分离蛋白中绿原酸引起的绿化

• DOI: 10.1016/j.foodres.2022.111996
• 发表时间: 2022
• 期刊: Food Research International
• 影响因子: 8.1
• 作者: Lo Verde, Christine;Pepra-Ameyaw, Nana Baah;Drucker, Charles T.;Okumura, Tracie L.S.;Lyon, Katherine A.;Muniz, Julia C.;Sermet, Chloe S.;Were Senger, Lilian;Owens, Cedric P.
• 通讯作者: Owens, Cedric P.