MECHANISTIC INVESTIGATION OF THE MYCOBACTERIAL GLYCOSYLTRANSFERASE GLFT2

分枝杆菌糖基转移酶 GLFT2 的机制研究

• 批准号: 8361170
• 负责人: Laura L Kiessling
• 金额: $ 0.14万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2012-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8361170
• 关键词: Active Sites; Anabolism; Antimycobacterial Agents; Biochemical; Biology; Carbohydrates; Cell Wall; Chemicals; Development; Disaccharides; Enzymes; Funding; Galactans; Galactose; Genus Mycobacterium; Glycobiology; Glycoconjugates; Grant; Hexoses; Investigation; Link; Microbe; Molecular; National Center for Research Resources; Parasites; Polymers; Polysaccharides; Positioning Attribute; Principal Investigator; Research; Research Infrastructure; Resources; Role; Site-Directed Mutagenesis; Source; Structure; Techniques; United States National Institutes of Health; Virulence; Work; arabinogalactan; base; cost; frontier; fungus; glycosyltransferase; insight; mycobacterial; novel; pyranose; small molecule; sugar

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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.

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 糖生物学，或研究碳水化合物的结构、生物合成和生物学是一个重要的研究前沿。六糖，如半乳糖，可以以吡喃糖或呋喃糖的形式存在。生物学上对吡喃糖的了解正在形成，但对含有呋喃糖的糖共轭化合物的了解相对较少。这些葡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)残留物对原生动物、寄生虫和真菌的毒力或生存能力至关重要。GALF因其在分枝杆菌中的作用而闻名，在分枝杆菌中，它是细胞壁的重要组成部分。分枝杆菌细胞壁的特征是一种基本的阿拉伯半乳聚糖聚合物。最近，糖基转移酶GlfT2的功能已被确定。这种酶负责聚合Galf残基，形成阿拉伯半乳糖的半乳糖多糖部分。自然产生的半乳糖链由交替的？-(1？5)和？-(1？6)Galf键组成。有趣的是，GlfT2负责这两个糖苷键的形成。目前还不清楚是什么支配着这种新的双功能活动。这项建议寻求使用化学和生化技术的组合来研究这种双功能的分子基础。定点突变将与？-(1？5)和？-(1？6)连接的GalF二糖受体底物一起使用，以确定GlfT2是使用一个还是两个活性位点来形成糖苷键。此外，还将合成阻止5-位或6-位伸长的氟化糖基受体和给体底物，并用于研究交替键的形成是否对GlfT2的活性是必需的。预计这些研究将提供对这种新的糖基转移酶活性的机械性见解，并为开发新的抗分枝杆菌小分子铺平道路。最近，参与分枝杆菌中半乳呋喃糖聚合物形成的糖基转移酶GlfT2的功能被确定。GlfT2催化形成两个不同的交替糖苷键。管理这一活动的机制尚不清楚。这项工作将调查支配这一双功能活动的因素。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。