MECHANISTIC INVESTIGATION OF THE MYCOBACTERIAL GLYCOSYLTRANSFERASE

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 糖生物学，或碳水化合物的结构，生物合成和生物学的研究是一个重要的研究前沿。 己糖，如半乳糖，可以吡喃糖或呋喃糖形式存在。 在生物学中对吡喃糖的理解正在出现，但对含呋喃糖的糖缀合物的了解相对较少。这些聚糖在各种微生物中的存在证明了它们的重要性。 呋喃半乳糖（Galf）残基对于原生动物寄生虫和真菌的毒力或存活力至关重要。 Galf以其在分枝杆菌中的作用而闻名，它是细胞壁的重要组成部分。 分枝杆菌细胞壁的特征在于必需的阿拉伯半乳聚糖聚合物。 最近，已经确定了糖基转移酶GlfT 2的功能。 该酶负责聚合Galf残基以形成阿拉伯半乳聚糖的半乳聚糖多糖部分。天然存在的半乳聚糖链由交替的？（1？5)然后呢？（1？6)Galf链接。 有趣的是，GlfT 2负责形成这两种糖苷键。 目前还不清楚是什么控制着这种新的双功能活动。 这项建议旨在调查这种双功能性的分子基础，使用化学和生物化学技术相结合。 定点突变将与？-（1？5)然后呢？（1？6)连接的Galf二糖受体底物，以确定GlfT 2是否使用一个或两个活性位点用于糖苷键形成。 此外，将合成排除5-或6-位延伸的氟化糖基受体和供体底物，并用于研究交替键的形成是否是GlfT 2活性的必需条件。 预计这些研究将提供这种新的糖基转移酶活性的机理洞察，并铺平了道路，走向新的抗分枝杆菌的小分子。最近建立的功能的糖基转移酶，GlfT 2，参与形成的半乳糖呋喃糖聚合物在分枝杆菌。 GlfT 2催化两个不同的交替糖苷键的形成。 控制这种活动的机制尚不清楚。 这项工作将调查的因素，支配这种双功能活动。 预计这些发现将有助于指导新的抗分枝杆菌化合物的开发。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Glycobiology, or the study of the structure, biosynthesis, and biology of carbohydrates is a significant research frontier. Hexoses, such as galactose, can exist in either the pyranose or furanose form. The understanding of pyranose sugars in biology is emerging, but comparatively less is known about furanose-containing glycoconjugates. The presence of these glycans in various microbes demonstrates their importance. Galactofuranose (Galf) residues are crucial for the virulence or viability of protozoan parasites and fungi. Galf is best known for its role in mycobacteria, where it is an essential component of the cell wall. The mycobacterial cell wall is characterized by an essential arabinogalactan polymer. Recently, the function of a glycosyltransferase, GlfT2, has been established. This enzyme is responsible for polymerizing Galf residues to form the galactan polysaccharide portion of arabinogalactan. Naturally occurring galactan chains consist of alternating ?-(1?5) and ?-(1?6) Galf linkages. Interestingly, GlfT2 is responsible for the formation of both of these glycosidic bonds. It is unclear what governs this novel bifunctional activity. This proposal seeks to investigate the molecular basis for this bifunctionality using a combination of chemical and biochemical techniques. Site-directed mutagenesis will be used in conjunction with ?-(1?5) and ?-(1?6) linked Galf disaccharide acceptor substrates to ascertain whether GlfT2 uses one or two active sites for glycosidic bond formation. In addition, fluorinated glycosyl acceptor and donor substrates that preclude elongation at either the 5- or 6-position will be synthesized and used to investigate whether the formation of alternating linkages is obligatory for activity of GlfT2. It is anticipated that these studies will provide mechanistic insight into this novel glycosyltransferase activity and pave the way towards new antimycobacterial small molecules.The function of a glycosyltransferase, GlfT2, involved in the formation of galactofuranose polymers in mycobacteria was recently established. GlfT2 catalyzes the formation of two distinct alternating glycosidic bonds. The mechanisms that govern this activity are not understood. This work will investigate the factors that govern this bifunctional activity. It is anticipated that these findings will help guide the development of new antimycobacterial compounds.