Riboflavinator: a molecular machine for efficient riboflavin biosynthesis

核黄素：高效核黄素生物合成的分子机器

• 批准号: 2204080
• 负责人: Audrey Lamb
• 金额: $ 48.61万
• 依托单位: University of Texas at San Antonio
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-12-15 至 2026-03-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2204080&HistoricalAwards=false
• 关键词: Riboflavinator; molecular; machine; efficient; riboflavin

With support from the Chemistry of Life Processes (CLP) Program in the Chemistry Division, Professor Audrey Lamb from the University of Texas at San Antonio (UTSA) will investigate the chemical mechanism of the biosynthesis of riboflavin (vitamin B2) with a special focus on the last enzymes in the pathway. Riboflavin is required by all living organisms and is synthesized from one guanosine 5’-triphosphate (GTP) and two ribulose 5-phosphate molecules using five unique enzymes. Despite unparalleled chemistry and the ubiquitous requirement for riboflavin, compelling evidence for the chemical mechanisms of the enzymes in this biosynthetic pathway has not been presented. We will investigate the hypothesis that the enzymes work in concert as a molecular machine for efficient production of this critical vitamin. This project will allow graduate students and postdoctoral fellows to acquire specialized training in enzyme kinetics and high-resolution X-ray crystallography. Portions of this project are structured such that undergraduate students will obtain meaningful research experiences to inspire careers in chemical research. This project also includes professional development outside of the laboratory for graduate students and postdoctoral fellows and pedagogical training for faculty at UTSA.Under this grant, the Lamb research team aims to establish how riboflavin is formed enzymatically from GTP and ribulose-5-phosphate, including structural identification of intermediates that are detected kinetically. This proposal focuses on the two final enzymes of the biosynthetic pathway that perform curious reactions. RibE (lumazine synthase) adds 4 carbons derived from the sugar ribulose 5-phosphate to a pyrimidinedione derived from GTP, generating lumazine. RibC performs an unusual dismutation reaction in which those same 4 carbons of the RibE reaction are transferred from one lumazine to another, generating riboflavin. The team will apply nuclear magnetic resonance methodology that they have developed to observe the formation and disappearance of intermediates for enzymes earlier in the biosynthetic pathway to substantiate or refute the largely hypothetical chemical mechanisms of RibE and RibC. Earlier work on this system has led the Lamb team to propose that efficient catalysis occurs when a molecular machine is formed, in which RibE forms as capsid that encapsulates the other 4 enzymes of the pathway. They have labelled this system the riboflavinator. The outcomes of this project are expected to advance the understanding of how this essential cofactor is biosynthesized.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.

在化学学部生命过程化学（CLP）项目的支持下，来自德克萨斯大学圣安东尼奥分校（UTSA）的奥黛丽·兰姆教授将研究核黄素（维生素B2）生物合成的化学机制，并特别关注该途径中的最后一种酶。核黄素是所有生物都需要的，由一个鸟苷5 ' -三磷酸（GTP）和两个5-磷酸核酮糖分子用五种独特的酶合成而成。尽管核黄素具有无与伦比的化学性质和无处不在的需求，但在这种生物合成途径中酶的化学机制尚未提出令人信服的证据。我们将研究这样一种假设，即酶作为一个分子机器协同工作，有效地生产这种关键的维生素。该项目将允许研究生和博士后获得酶动力学和高分辨率x射线晶体学方面的专业培训。本项目的部分内容旨在使本科生获得有意义的研究经验，以激发化学研究的职业生涯。该项目还包括实验室以外的研究生和博士后的专业发展，以及对UTSA教师的教学培训。在这项资助下，Lamb研究小组的目标是确定GTP和5-磷酸核酮糖是如何通过酶催化形成核黄素的，包括对动力学检测的中间体进行结构鉴定。这一建议侧重于生物合成途径的最后两种酶，它们执行奇怪的反应。RibE （lumazine synthase）将5-磷酸核酮糖衍生的4个碳添加到GTP衍生的嘧啶二酮上，生成lumazine。RibC进行了一种不寻常的突变反应，RibE反应中相同的4个碳从一种嘧啶转移到另一种嘧啶，产生核黄素。该团队将应用他们开发的核磁共振方法来观察生物合成途径早期酶中间体的形成和消失，以证实或反驳RibE和RibC的主要假设的化学机制。Lamb团队在该系统的早期工作中提出，当分子机器形成时，有效的催化作用就会发生，在这个分子机器中，RibE作为衣壳形成，包裹了该途径的其他4种酶。他们把这个系统称为核黄素发生器。该项目的结果有望促进对这种重要辅因子如何生物合成的理解。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。