APPLICATION OF NMR TO THE STUDY OF NDP-SUGAR BIOSYNTHESIS

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。作为阐明植物和人类病原体新型隐球菌中所有核苷酸-糖合成的计划的一部分，我们在过去几年中鉴定了参与UDP-木糖、UDP-芹菜糖、UDP-葡萄糖醛酸、UDP-鼠李糖等合成的生物合成基因。NMR用于鉴定每种重组蛋白的酶促产物。我们还试图使用NMR直接研究酶-底物系统，以避免色谱步骤和由于化学不稳定性而导致的材料损失。这些研究包括鉴定不同孵育时间期间的酶中间体，确定这些反应的酶动力学，以及随后的产物降解。 所提供的服务：收集来自酶制剂的各种UDP-糖的质子NMR光谱。这些光谱的持续分析表明存在所需产物，如UDP-葡萄糖醛酸和UDP-木糖。其他目标，如UDP-apiose，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.