The Chemistry and Biology of Galactofuranose Residues

描述（由申请人提供）：拟议研究的总体目标是探索半乳糖呋喃糖（Galf）残基的生物合成掺入。半胱氨酸残基存在于许多病原体中，但在哺乳动物中不存在。在本应用中，我们重点了解和抑制两种参与含有半乳糖残基的糖缀合物生物合成的酶：黄酶udp -半乳糖氨基葡萄糖mutase （UGM）和假定的半乳糖呋喃基转移酶GlfT。这两种酶都参与分枝杆菌细胞壁的生物合成，并且都对结核分枝杆菌（结核的病原体）的生存能力至关重要。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仍然存在许多问题。例如，UGM催化udp -半乳糖醛酸糖（UDP-Galp）和udp -半乳糖呋喃糖（UDP-Galf）异构化的机制尚不清楚。关于GlfT的了解更少，它似乎将半乳糖呋喃糖残基转移到脂质前体。拟议的研究有三个目的。第一个目的是验证我们的假设，即UDP- Galp和UDP- galf的异构化是通过N(5)黄素衍生的铝离子进行的。这种黄素催化模式以前没有被观察到，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是利用荧光偏振法鉴定UGM抑制剂。该目的的一个目标是鉴定分子支架，从中可以生成组合文库以优化效力。在目标3中，我们建议研究GlfT。目的是开发一种有效的方法来产生这种假定的糖基转移酶，并表征其底物特异性。

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.