Rafty Louise A, Schmidt Manning T, Perraud Anne-Laure, Scharenberg Andrew M, Denu John M
Oregon Health & Science University, Department of Biochemistry and Molecular Biology, Portland, Oregon 97201-3098, USA.
J Biol Chem. 2002 Dec 6;277(49):47114-22. doi: 10.1074/jbc.M208997200. Epub 2002 Oct 4.
The Sir2 family of NAD(+)-dependent histone/protein deacetylases has been implicated in a wide range of biological activities, including gene silencing, life span extension, and chromosomal stability. Recent evidence has indicated that these proteins produce a novel metabolite O-acetyl-ADP-ribose (OAADPr) during deacetylation. Cellular studies have demonstrated that this metabolite exhibits biological effects when microinjected in living cells. However, the molecular targets of OAADPr remain to be identified. Here we have analyzed the ADP-ribose-specific Nudix family of hydrolases as potential in vivo metabolizing enzymes of OAADPr. In vitro, we found that the ADP-ribose hydrolases (yeast YSA1, mouse NudT5, and human NUDT9) cleaved OAADPr to the products AMP and acetylated ribose 5'-phosphate. Steady-state kinetic analyses revealed that YSA1 and NudT5 hydrolyzed OAADPr with similar kinetic constants to those obtained with ADP-ribose as substrate. In dramatic contrast, human NUDT9 was 500-fold less efficient (k(cat)/K(m) values) at hydrolyzing OAADPr compared with ADP-ribose. The inability of OAADPr to inhibit the reaction of NUDT9 with ADP-ribose suggests that NUDT9 binds OAADPr with low affinity, likely due to steric considerations of the additional acetylated-ribose moiety. We next explored whether Nudix hydrolytic activities against OAADPr could be observed in cell extracts from yeast and human. Using a detailed analysis of the products generated during the consumption of OAADPr in extracts, we identified two robust enzymatic activities that were not consistent with the known Nudix hydrolases. Instead, we identified cytoplasmic esterase activities that hydrolyze OAADPr to acetate and ADP-ribose, whereas a distinct activity residing in the nucleus is consistent with an OAADPr-specific acetyltransferase. These findings establish for the first time that select members of the ADP-ribose hydrolases are potential targets of OAADPr metabolism. However, the predominate endogenous activities observed from diverse cell extracts represent novel enzymes.
NAD⁺依赖的组蛋白/蛋白质去乙酰化酶Sir2家族参与了广泛的生物学活动，包括基因沉默、寿命延长和染色体稳定性。最近的证据表明，这些蛋白质在去乙酰化过程中产生一种新的代谢产物O-乙酰-ADP-核糖（OAADPr）。细胞研究表明，这种代谢产物在微注射到活细胞中时具有生物学效应。然而，OAADPr的分子靶点仍有待确定。在这里，我们分析了ADP-核糖特异性的Nudix水解酶家族，作为OAADPr潜在的体内代谢酶。在体外，我们发现ADP-核糖水解酶（酵母YSA1、小鼠NudT5和人类NUDT9）将OAADPr裂解为产物AMP和乙酰化核糖5'-磷酸。稳态动力学分析表明，YSA1和NudT5水解OAADPr的动力学常数与以ADP-核糖为底物时获得的相似。形成鲜明对比的是，与ADP-核糖相比，人类NUDT9水解OAADPr的效率低500倍（kcat/Km值）。OAADPr无法抑制NUDT9与ADP-核糖的反应，这表明NUDT9与OAADPr的结合亲和力较低，可能是由于额外的乙酰化核糖部分的空间位阻。接下来，我们探讨了在酵母和人类的细胞提取物中是否能观察到针对OAADPr的Nudix水解活性。通过对提取物中OAADPr消耗过程中产生的产物进行详细分析，我们鉴定出两种强大的酶活性，这与已知的Nudix水解酶不一致。相反，我们鉴定出细胞质酯酶活性，它将OAADPr水解为乙酸盐和ADP-核糖，而存在于细胞核中的一种独特活性与一种OAADPr特异性乙酰转移酶一致。这些发现首次证实，ADP-核糖水解酶的某些成员是OAADPr代谢的潜在靶点。然而，从不同细胞提取物中观察到的主要内源性活性代表了新的酶。
