The Chemistry and Biology of Galactofuranose Residues

描述（由申请人提供）：半乳糖呋喃糖（Galf）残基与包括分枝杆菌在内的许多微生物的毒力或活力有关。本研究的目的是了解半胱氨酸残基与分枝杆菌细胞壁结合的机制。我们将研究这一过程中两个关键酶的结构、催化机制和功能：尿苷-5'-二磷酸(UDP)-半乳糖氨基糖mutase （Glf或UGM）和半乳糖基呋喃基转移酶GlfT2。以下是该应用程序的三个特定目标。目的1是了解黄酶UGM的作用机制。阐明UGM的催化机制将增强我们对黄酮类酶的多种化学性质的理解，为细胞壁生物合成的化学基础提供见解，并指导这种必需酶抑制剂的产生。目的2是产生有效的和细胞渗透性的UGM抑制剂，可以用作细胞壁生物合成的探针，并作为开发新的抗细菌药物的先导。目的3是研究GlfT2酶，该酶催化半乳糖聚合物的合成，该聚合物由1,5-和1,6-连接的半乳糖残基交替组成。我们将测试聚合是否是过程性的，探索单个酶如何产生两个区域异构体糖键，并确定如何控制聚合物长度。这些研究将阐明分枝杆菌中半乳糖生物合成和多糖生物合成的机制。为了实现这些目标，我们将采用来自有机化学、糖生物学、碳水化合物化学、化学酶学、结构生物学、微生物学和化学生物学的方法和思想。意义：本研究的结果将为分枝杆菌细胞壁的重要组成部分——半乳聚糖聚合物的组装提供新的见解。他们还将解决生物系统如何在没有模板的情况下控制聚合物长度的基本问题。这些知识将应用于开发阻断分枝杆菌细胞生长的小分子。这些试剂将作为分枝杆菌细胞壁生物合成的有价值的探针，并为开发新的抗分枝杆菌药物提供线索。

DESCRIPTION (provided by applicant): Galactofuranose (Galf) residues have been implicated in the virulence or viability of many microbes, including mycobacteria. The goal of the proposed research is to understand the mechanisms underlying Galf residue incorporation into the mycobacterial cell wall. We shall investigate the structure, catalytic mechanism, and function of two key enzymes in this process: the flavoenzyme uridine-5'-diphosphate (UDP)-galactopyranose mutase (Glf or UGM) and the galactosylfuranosyltransferase GlfT2. The three Specific Aims of this application follow. Aim 1 is to understand the mechanism of the flavoenzyme UGM. Elucidating the catalytic mechanism UGM will enhance our understanding of the diverse chemistry of the flavoenzymes, provide insight into the chemistry underlying cell wall biosynthesis, and guide the generation of inhibitors of this essential enzyme. Aim 2 is to generate potent and cell-permeable inhibitors of UGM that can be used as probes of cell wall biosynthesis and as leads for the development of new antimycobacterial agents. Aim 3 is to investigate the enzyme GlfT2, which catalyzes the synthesis of a galactan polymer composed of alternating 1,5- and 1,6-linked Galf residues. We shall test whether the polymerization is processive, explore how a single enzyme generates two regioisomeric sugar linkages, and determine how polymer length is controlled. These investigations will illuminate the mechanisms underlying galactan biosynthesis in mycobacteria and the biosynthesis of polysaccharides, in general. In pursuing these Aims, we shall employ methods and ideas from organic chemistry, glycobiology, carbohydrate chemistry, chemical enzymology, structural biology, microbiology, and chemical biology. Significance: The results of the proposed research will provide new insights into the assembly of the galactan polymer, an essential component of the mycobacterial cell wall. They also will address the fundamental question of how biological systems control polymer length in the absence of a template. This knowledge will be applied to develop small molecules that block mycobacterial cell growth. Such agents will serve as valuable probes of mycobacterial cell wall biosynthesis and as leads for the development of new antimycobacterial drugs. PUBLIC HEALTH RELEVANCE: This research project is focused on understanding essential steps in the biosynthesis of the mycobacterial cell wall. Mycobacteria cause a number of diseases, including tuberculosis (TB). TB causes about 1.7 million deaths each year, and current therapies are failing. The goal of this project is to understand key steps in mycobacterial cell wall biosynthesis that are not targeted by any current drugs and find inhibitors from which new types of drugs could be developed.