THE CHEMISTRY AND BIOLOGY OF GALACTOFURANOSE

呋喃半乳糖的化学和生物学

• 批准号: 8168936
• 负责人: Laura L Kiessling
• 金额: $ 0.03万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-01 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8168936
• 关键词: Active Sites; Amino Acids; Bacteria; Binding; Binding Sites; Biology; Cell Wall; Chemistry; Computer Retrieval of Information on Scientific Projects Database; Drug Delivery Systems; Enzymes; Excision; Flavins; Funding; Galactose; Grant; Institution; Label; Mammalian Cell; Molecular Weight; Mutate; Mycobacterium tuberculosis; Oxidation-Reduction; Protocols documentation; Research; Research Personnel; Resources; Role; Source; UDP-galactopyranose mutase; United States National Institutes of Health; adduct; cofactor; uridine diphosphate galactofuranose

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. The flavoenzyme UDP-galactopyranose mutase (UGM) catalyzes the interconversion of UDP-galactopyranose and UDP-galactofuranose. Galactofuranose residues, which are not found in mammalian cells, are then incorporated into the cell walls of certain bacteria, including Mycobacterium tuberculosis, making UGM an attractive drug target. UGM is a flavoenzyme, but the precise role of the cofactor has been unclear due to the lack of reduction/oxidation chemistry in this isomerization. We propose that turnover occurs by a covalent flavin-galactose iminium species. Using NaCNBH3 to trap this adduct, analysis of the resulting species by LCMS shows a peak corresponding to the molecular weight of the proposed alkylated flavin, as well as an absorbance spectrum indicative of an N(5) alkylflavin. In order to investigate how the enzyme allows for this unique chemistry, we have begun to mutate conserved residues around the active site in order to establish the mechanistic roles of certain amino acids. A protocol for removal of the noncovalently bound flavin and replacement with isotopically labeled flavin has been developed to allow for NMR studies of the flavin binding site, as well as to use NMR to visualize the connectivities of the covalent alkylflavin.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 Flavoenzyme UDP - 半乳吡喃糖突变酶（UGM）催化了UDP-半乳吡喃糖和UDP-乳乳凝糖酶的相互转换。 然后在哺乳动物细胞中找不到的半乳呋喃糖残基被掺入某些细菌的细胞壁中，包括结核分枝杆菌，使UGM成为有吸引力的药物靶标。 UGM是一种黄酮酶，但是由于这种异构化中缺乏还原/氧化化学，辅因子的确切作用尚不清楚。 我们建议通过共价黄乳糖亚基氨基属物种发生营业额。 使用NaCNBH3捕获这种加合物，LCMS对所得物种的分析显示了与所提出的烷基化黄素分子量相对应的峰，以及指示N（5）烷基氟法素的吸光度光谱。 为了研究酶如何允许这种独特的化学作用，我们已经开始在活性位点上突变保守的残基，以确定某些氨基酸的机械作用。 已经开发了一种用于去除非共价结合黄素并用同位素标记黄素替代的方案，以允许对黄素结合位点进行NMR研究，并使用NMR可视化共价烷基氟法的连接性。