APPLICATION OF NMR TO THE STUDY OF NDP-SUGAR BIOSYNTHESIS

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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.