STUDIES OF NAD+-DEPENDENT DEACETYLATION AND NAD+ BIOSYNTHETIC ENZYMES

点击翻译按钮获取中文摘要

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. We are requesting beamtime for two related projects in the lab: mechanistic studies of NAD+-dependent deacetylation by sirtuins, and studies of substrate binding to a mammalian NAD+ biosynthetic enzyme. Sir2 enzyme catalyze the NAD+-dependent deacetylation of lysine residues in a two-step mechanism that includes cleavage of nicotinamide from NAD+, followed by transfer of the acetyl group to ADP ribose. While a number of sirtuin structures have been determined, remaining questions center on the precise mechanism of enzymatic reaction. We have devised schemes to trap the reaction O-alkyl amidate intermediate in the crystals and wish to collect data on these crystals. One approach is to use a thio-acetylated peptide, which forms a long-lived covalent complex with ADP ribose. A second approach is to use a mutant enzyme defective in the second step of the reaction, and to soak NAD+ into crystals containing enzyme bound to peptide. Crystals are in hand for all of these. The second project is to continue structural studies of Nampt (nicotinamide phosphoribosyltransferase), whose structure we recently solved in complex with NMN, a product of the reaction. We now wish to determine structures of the enzyme bound to substrate, as well as of the phosphorylated form of the enzyme.