CAREER: Defining the chemical contributions of Val29 and Tyr30 in Mycofactocin Biosynthesis for the Development of Novel Redox Molecules.

在化学系生命过程化学（CLP）项目的支持下，丹佛大学的John A. Latham博士将研究细菌维生素分枝杆菌素合成过程中的化学过程。细菌产生许多化合物或天然产物，使资源竞争、解毒环境和执行关键的细胞过程成为可能。分枝杆菌素，一种细菌维生素，在分枝杆菌的关键细胞过程中需要穿梭电子。分枝杆菌素是由一种使用强大酶MftC的肽合成的，与其他肽天然产物相比，这种方式是不寻常的。这项研究有望导致对这种化学的新认识和一类新的生物工程氧化还原辅助因子，这反过来将有助于阐明分枝因子素依赖的生理过程。这项工作使研究生和中学科学教师在机械酶学和理性生物工程方面获得专门的训练。此外，通过外展计划，这项工作将纳入高中和初中学生制作的产品。分枝杆菌素成熟酶（MftC）是一种自由基s -腺苷甲硫氨酸酶（rSAM），它催化了分枝杆菌素生物合成中最关键的步骤。它只是自然界中广泛的自由基SAM酶中的一种，因此在这个项目中收集的机制见解可能会对这一重要酶类的其他成员的研究和理解产生更广泛的科学影响。MftC通过氧化脱羧c端酪氨酸和在倒数第二缬氨酸和c端酪氨酸之间形成内酰胺来修饰前体肽MftA。将评估MftC的反应范围，以确定c端电子汇（酪氨酸）的需求，以及缬氨酸的保存是否调节了分枝杆菌素的中点电位。目的1通过评价酪氨酸类似物的动力学同位素效应和pKa效应来确定酪氨酸的机制作用。目的2通过评价取代基对分枝杆菌素中点电位的影响，建立保存缬氨酸的化学逻辑。该项目将促进对rSAM机制的理解，并为合理设计一类新的氧化还原辅助因子建立框架。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。

With the support from the Chemistry of Life Processes (CLP) Program in the Division of Chemistry (CHE), Dr. John A. Latham from the University of Denver will investigate how the chemistry is carried out in the synthesis of the bacterial vitamin, mycofactocin. Bacteria produce numerous compounds, or natural products that enable competition for resources, detoxifying environments, and carrying out critical cellular processes. Mycofactocin, a bacterial vitamin which is required for shuttling electrons in critical cellular processes in Mycobacteria. Mycofactocin is made from a peptide using a powerful enzyme, MftC, in a way that is unusual in comparison to other peptide natural products. This study is expected to result in new understanding of this chemistry and a new class of bioengineered redox cofactors, which in turn will help to elucidate mycofactocin-dependent physiological processes. This work allows for graduate students and middle school science teachers to gain specialized training in mechanistic enzymology and rational bioengineering. In addition, through outreach programming, this work will incorporate products produced by high school and middle school students. Mycofactocin maturase, MftC, is a radical S-adenosylmethionine enzyme (rSAM) that catalyzes the most critical steps in mycofactocin biosynthesis. It is just one of a broad class of radical SAM enzymes in Nature and so mechanistic insights gleaned in this project will likely have much broader scientific impact upon studies and understanding of other members of this important enzymatic class. MftC modifies the precursor peptide, MftA, by oxidatively decarboxylating the C-terminal tyrosine and by the formation of a lactam between the penultimate valine and the C-terminal tyrosine. The reaction scope of MftC will be evaluated to determine the requirement of an electron sink (tyrosine) on the C-terminus and if the conservation of valine tunes the midpoint potential of mycofactocin. Objective 1 focuses on defining the mechanistic role of tyrosine by evaluating kinetic isotope effects and pKa effects of tyrosine analogs. Objective 2 establishes the chemical logic for the conservation of valine by evaluating substituent effects on the midpoint potential of mycofactocin. The project will advance the understanding rSAM mechanisms and establish the framework for the rational design of a new class of redox 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.