The Chemistry and Biology of Galactofuranose Residues

描述（由申请方提供）：拟定研究的总体目标是探索呋喃半乳糖（Galf）残基的生物合成掺入。Galf残基存在于许多病原体中，但它们不存在于哺乳动物中。在这个应用程序中，我们专注于了解和抑制两种酶参与含半乳糖醛酸残基的糖缀合物的生物合成：黄素酶UDP-吡喃半乳糖苷酶（UGM）和推定的半乳糖呋喃糖基转移酶GlfT。这两种酶都参与分枝杆菌细胞壁的生物合成，并且都是结核病病原体结核分枝杆菌生存所必需的。尽管它们作为治疗靶点具有潜在的重要性，但关于这些酶如何参与分枝杆菌细胞壁的生物合成的许多问题仍然存在。例如，UGM催化UDP-吡喃半乳糖（UDP-Galp）和UDP-呋喃半乳糖（UDP-Galf）异构化的机制尚不清楚。关于GlfT的了解甚至更少，GlfT似乎将半乳糖呋喃糖残基转移到脂质前体。这项研究有三个目的。第一个目的是集中于测试我们的假设，即UDP-Galp和UDP-Galf的异构化通过N（5）黄素衍生的亚胺离子进行。这种黄素催化的模式以前没有观察到，目的1中提出的实验旨在评估这种机制建议的可行性。目的2是针对使用荧光偏振测定来鉴定UGM的抑制剂。该目的的一个目标是鉴定分子支架，从该分子支架可以产生组合文库以优化效力。在目标3中，我们建议研究GlfT。我们的目标是开发一种有效的方法来产生这种推定的糖基转移酶，并表征其底物特异性。 我们预计，这里提出的研究将为调查这个有趣的碳水化合物单位的作用开辟新的途径。此外，我们希望，通过开展拟议的调查，可能会出现治疗结核病的新线索。

DESCRIPTION (provided by applicant): The overall objective of the proposed research is to explore the biosynthetic incorporation of galactofuranose (Galf) residues. Galf residues are found in many pathogens, but they are not present in mammals. In this application, we focus on understanding and inhibiting two enzymes involved in the biosynthesis of glycoconjugates containing Galf residues: the flavoenzyme UDP-galactopyranose mutase (UGM) and the putative galactofuranosyltransferase GlfT. Both of these enzymes are involved in mycobacterial cell wall biosynthesis, and both are essential for the viability of Mycobacterium tuberculosis, the causative agent of tuberculosis. Despite their potential importance as therapeutic targets, many questions regarding how these enzymes participate in biosynthesis of the mycobacterial cell wall remain. For example, the mechanism by which UGM catalyzes the isomerization of UDP-galactopyranose (UDP-Galp) and UDP-galactofuranose (UDP-Galf) is unknown. Even less is known about GlfT, which appears to transfer galactofuranose residues to lipid precursors. The proposed research has three aims. The first aim is focused on testing our hypothesis that the isomerization of UDP- Galp and UDP-Galf proceeds via an N(5) flavin-derived iminium ion. This mode of flavin catalysis has not been observed previously/and the experiments are proposed in aim 1 are designed to evaluate the feasibility of this mechanistic proposal. Aim 2 is directed at identifying inhibitors of UGM using a fluorescence polarization assay. One goal of this aim is to identify molecular scaffolds from which combinatorial libraries can be generated to optimize potency. In aim 3, we propose to investigate GlfT. The goals are to develop an effective method to produce this putative glycosyltransferase and to characterize its substrate specificity. We anticipate that the studies proposed here will open new avenues for investigating the roles of this interesting carbohydrate unit. Moreover, we hope that by pursuing the proposed investigations, new leads for the treatment of tuberculosis may emerge.