CAREER: Molecular Evolution of Oxidant Specificity in Bacterial Flavoprotein Amine Oxidases

职业：细菌黄素蛋白胺氧化酶氧化特异性的分子进化

• 批准号: 2236541
• 负责人: Frederick Stull
• 金额: $ 75.7万
• 依托单位: Western Michigan University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2028-06-30
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2236541&HistoricalAwards=false
• 关键词: CAREER; Molecular; Evolution; Oxidant; Specificity

With this CAREER award, supported by the Chemistry of Life Processes Program in the Division of Chemistry, Frederick Stull from Western Michigan University is studying the evolution of oxidant specificity in bacterial flavoprotein amine oxidases. Flavoprotein amine oxidases are enzymes that catalyze critical reactions throughout cellular metabolism, and most are thought to transfer electrons from their organic substrates to molecular oxygen. It was recently discovered that certain bacterial flavoprotein amine oxidase enzymes break this mold by transferring electrons from their substrates to cytochrome c proteins instead of directly to molecular oxygen. This project is evaluating if this use of cytochrome c is more widespread among bacterial flavoprotein amine oxidases than previously realized and seeks to understand how these divergent functions evolved in this widespread enzyme family. The basic science knowledge obtained from this project could be used to achieve reactions that are useful for biotechnology. The graduate and undergraduate students involved in this project will gain hands-on experience in spectroscopy, transient kinetics and enzyme engineering. Selected enzymes from this project are to be used to develop a hypothesis-driven laboratory course for undergraduate students that directly contributes to the scientific goals of this project. This project will also include development of a platform for facilitating the engagement of undergraduate work-study recipients in hands-on research. Frederick Stull and his team have recently discovered that nicotine oxidoreductase and pseudooxynicotine amine oxidase, both members of the flavoprotein amine oxidase (FAO) superfamily, are not actually oxidases. These two enzymes transfer electrons from their substrates to a cytochrome c protein instead of O2, meaning that they are dehydrogenases. This project will test the hypothesis that many other bacterial flavoprotein amine oxidases also use cytochrome c as an oxidant instead of O2, severely undermining the oxidase paradigm ascribed to the FAO enzyme family. The oxidant specificity of select bacterial FAOs that are predicted to be cytochrome c-utilizing dehydrogenases, as well as closely related oxidases, will be characterized by transient kinetics. Experimental evolution, phylogenetic analysis and ancestral reconstruction will be used to identify the structural features that dictate flavin-oxidant reactivity in bacterial FAOs. Information from this study will likely provide new insight into how flavoenzymes control the reactivity of their flavin cofactors.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.

有了这个职业奖，由化学部生命过程计划的化学支持，来自西密歇根大学的Frederick Stull正在研究细菌黄素蛋白胺氧化酶中氧化剂特异性的演变。黄素蛋白胺氧化酶是催化整个细胞代谢的关键反应的酶，并且大多数被认为将电子从其有机底物转移到分子氧。最近发现，某些细菌黄素蛋白胺氧化酶通过将电子从其底物转移到细胞色素c蛋白而不是直接转移到分子氧来打破这种模式。该项目正在评估细胞色素c在细菌黄素蛋白胺氧化酶中的使用是否比以前认识到的更广泛，并试图了解这些不同的功能是如何在这个广泛的酶家族中进化的。从该项目中获得的基础科学知识可用于实现对生物技术有用的反应。参与该项目的研究生和本科生将获得光谱学，瞬态动力学和酶工程方面的实践经验。从这个项目中选择的酶将用于开发一个假设驱动的实验室课程，直接有助于本项目的科学目标的本科生。该项目还将包括开发一个平台，以促进本科半工半读学生参与实践研究。 Frederick Stull和他的团队最近发现，尼古丁氧化还原酶和假氧化尼古丁胺氧化酶，都是黄素蛋白胺氧化酶（FAO）超家族的成员，实际上不是氧化酶。这两种酶将电子从其底物转移到细胞色素c蛋白而不是O2，这意味着它们是脱氢酶。该项目将测试许多其他细菌黄素蛋白胺氧化酶也使用细胞色素c作为氧化剂而不是O2的假设，严重破坏了归因于FAO酶家族的氧化酶范例。氧化剂特异性的选择细菌FAOs预测为细胞色素c利用氧化酶，以及密切相关的氧化酶，其特征在于瞬态动力学。实验进化，系统发育分析和祖先重建将被用来确定的结构特征，决定黄素氧化反应在细菌FAOs。这项研究的信息可能会为黄素酶如何控制其黄素辅因子的反应性提供新的见解。该奖项反映了NSF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。