APPLICATION OF NMR TO THE STUDY OF NDP-SUGAR BIOSYNTHESIS

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。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，我们在过去几年中已经确定了参与合成udp -木糖，UDP-apiose， udp -葡萄糖醛酸，udp -鼠李糖等的生物合成基因。核磁共振用于鉴定每个重组蛋白的酶促产物。我们也在尝试使用核磁共振直接研究酶-底物体系，以避免色谱步骤和由于化学不稳定性造成的材料损失。这些研究包括在不同的孵育时间内鉴定酶的中间体，确定这些反应的酶动力学，以及跟踪产物的降解。所执行的服务：收集了各种来自酶制剂的udp -糖的质子核磁共振光谱。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。其他目标，如民主统一党，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.