APPLICATION OF NMR TO THE STUDY OF NDP-SUGAR BIOSYNTHESIS

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。作为阐明植物和人类病原菌新生隐球菌中所有核苷酸-糖合成的计划的一部分，我们在过去的几年中已经鉴定了参与UDP-木糖、UDP-apiose、UDP-葡萄糖醛酸、UDP-鼠李糖等合成的生物合成基因。核磁共振用于鉴定每个重组蛋白的酶产物。我们还试图利用核磁共振技术直接研究酶-底物体系，以避免由于化学不稳定而造成的层析步骤和物质的损失。这些研究包括确定不同孵化时间的酶中间体，确定这些反应的酶动力学，以及产物的后续降解。提供的服务：收集了从酶制剂中提取的各种UDP-糖的质子核磁共振谱。对这些光谱的持续分析表明，存在所需的产物，如UDP-葡萄糖醛酸和UDP-木糖。UDP-apiose等其他目标仍在调查中。

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. As part of a program to elucidate the synthesis of all nucleotide-sugars in plants and in the human pathogen Cryptococcus neoformans, we have identified in the past several years biosynthetic genes involved in synthesis of UDP-xylose, UDP-apiose, UDP-glucuronic acid, UDP-rhamnose and others. NMR is used to identify the enzymatic product of each recombinant protein. We also are trying to use NMR to directly study the enzyme-substrate systems in order to avoid chromatographic steps and loss of material due to chemical instability. These studies include identifying the enzymatic intermediates during different incubation times, determining enzyme kinetics of these reactions, and following degradation of products. Service performed: Proton NMR spectra were collected on a variety of UDP-sugars derived from enzyme preparations. The ongoing analysis of these spectra indicates the presence of the desired products such as UDP-glucuronic acid and UDP-xylose. Other targets such as UDP-apiose are still being investigated.