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呋喃葡萄糖(Galf)残基的生物合成并入。GALF残留物存在于许多病原体中，但在哺乳动物中不存在。在这一应用中，我们重点了解和抑制与含有GalF残基的糖偶联物生物合成有关的两种酶：黄素酶UDP-半乳糖苷变位酶(UGM)和推测的半乳糖呋喃糖基转移酶GlfT。这两种酶都参与了分枝杆菌细胞壁的生物合成，对结核分枝杆菌的生存至关重要，而结核分枝杆菌是结核病的病原体。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-半乳糖(UDP-Galp)和UDP-半乳呋喃糖(UDP-Galf)异构化的机理尚不清楚。对谷氨酰转氨酶的了解更少，它似乎将半乳呋喃糖残基转移到脂质前体。这项拟议的研究有三个目标。第一个目标是验证我们的假设，即UDP-Galp和UDP-Galf的异构化是通过N(5)黄素衍生的亚胺离子进行的。这种黄素催化模式以前没有观察到过，在目标1中提出了实验，目的1是为了评估这一机械方案的可行性。目的2旨在利用荧光偏振分析鉴定UGM的抑制剂。这一目标的一个目标是识别分子支架，从这些支架上可以产生组合文库，以优化效力。在目标3中，我们建议对Glft进行研究。我们的目标是开发一种有效的方法来生产这种假定的糖基转移酶，并表征其底物特异性。 我们预计，这里提出的研究将为研究这一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.