Riboflavinator: a molecular machine for efficient riboflavin biosynthesis

在化学系生命过程化学（CLP）项目的支持下，德克萨斯大学圣安东尼奥分校（UTSA）的Audrey Lamb教授将研究核黄素（维生素B2）生物合成的化学机制，特别关注途径中的最后一种酶。 核黄素是所有生物体所必需的，并且使用五种独特的酶从一个鸟苷5 '-三磷酸（GTP）和两个核酮糖5-磷酸分子合成。尽管无与伦比的化学和无处不在的核黄素的需求，在这个生物合成途径的酶的化学机制还没有令人信服的证据。 我们将研究的假设，酶协同工作，作为一个分子机器，有效地生产这种关键的维生素。 该项目将使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。 该项目的部分结构，使本科生将获得有意义的研究经验，激发化学研究的职业生涯。该项目还包括研究生和博士后研究员的实验室外专业发展以及UTSA教师的教学培训。在此资助下，Lamb研究团队旨在确定核黄素是如何从GTP和核酮糖-5-磷酸酶促形成的，包括动力学检测的中间体的结构鉴定。这一建议集中在生物合成途径的两个最后的酶，执行奇怪的反应。 里贝（lumazine synthase）将来自糖核酮糖5-磷酸的4个碳添加到来自GTP的嘧啶二酮，产生lumazine。RibC进行一种不寻常的歧化反应，其中里贝反应的相同4个碳从一个Lumazine转移到另一个，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，以证实或反驳里贝和RibC的主要假设化学机制。 关于这个系统的早期工作已经导致Lamb团队提出，当分子机器形成时，有效的催化作用就会发生，其中里贝形成为衣壳，封装了该途径的其他4种酶。 他们把这个系统称为核黄素转运子。 该项目的成果有望促进对这种重要辅因子是如何生物合成的理解。该奖项反映了NSF的法定使命，并被认为值得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来支持。

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.