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

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。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，但不一定是研究者所在的机构。 我们正在为实验室中的两个相关项目请求beamtime：sirtuins的NAD+依赖性脱乙酰化的机制研究，以及底物与哺乳动物NAD+生物合成酶结合的研究。Sir 2酶以两步机制催化赖氨酸残基的NAD+依赖性脱乙酰化，包括从NAD+裂解烟酰胺，然后将乙酰基转移到ADP核糖。虽然一些sirtuin结构已被确定，剩余的问题集中在酶促反应的精确机制。我们已经设计了方案，以捕获反应O-烷基酰胺中间体的晶体，并希望收集这些晶体上的数据。一种方法是使用硫代乙酰化肽，其与ADP核糖形成长寿命的共价复合物。第二种方法是使用在反应的第二步中有缺陷的突变酶，并将NAD+浸泡到含有与肽结合的酶的晶体中。所有这些都有水晶在手。第二个项目是继续Nampt（烟酰胺磷酸核糖基转移酶）的结构研究，我们最近在与NMN（反应产物）的复合物中解决了Nampt的结构。我们现在希望确定与底物结合的酶的结构，以及酶的磷酸化形式。

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.