THE CHEMISTRY AND BIOLOGY OF GALACTOFURANOSE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 黄素酶UDP-半乳糖变位酶(UGM)催化UDP-半乳糖和UDP-呋喃半乳糖的相互转化。在哺乳动物细胞中没有发现的呋喃半乳糖残留物，然后被结合到包括结核分枝杆菌在内的某些细菌的细胞壁中，使UGM成为一个有吸引力的药物靶点。UGM是一种黄素酶，但由于在这种异构化过程中缺乏还原/氧化化学，辅助因子的确切作用尚不清楚。我们认为周转是通过共价的黄素-半乳糖亚胺物种发生的。用NaCNBH3捕获这种加合物，用LC MS对所得物种进行分析，结果显示一个与所提议的烷基化黄素的分子量相对应的峰，以及一个指示N(5)烷基黄素的吸收光谱。为了研究酶是如何允许这种独特的化学作用的，我们已经开始突变活性部位周围的保守残基，以建立某些氨基酸的机制作用。已经开发了一种用于去除非共价结合的黄素并用同位素标记的黄素取代的方案，以允许对黄素结合部位的核磁共振研究，以及使用核磁共振来可视化共价烷基黄素的连接性。

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.