Silent information regulator 2 (Sir2) enzymes catalyze NAD؉ -dependent protein/histone deacetylation, where the acetyl group from the lysine ⑀-amino group is transferred to the ADP-ribose moiety of NAD ؉ , producing nicotinamide and the novel metabolite O-acetyl-ADPribose. Sir2 proteins have been shown to regulate gene silencing, metabolic enzymes, and life span. Recently, nicotinamide has been implicated as a direct negative regulator of cellular Sir2 function; however, the mechanism of nicotinamide inhibition was not established. Sir2 enzymes are multifunctional in that the deacetylase reaction involves the cleavage of the nicotinamide-ribosyl, cleavage of an amide bond, and transfer of the acetyl group ultimately to the 2-ribose hydroxyl of ADP-ribose. Here we demonstrate that nicotinamide inhibition is the result of nicotinamide intercepting an ADP-ribosyl-enzyme-acetyl peptide intermediate with regeneration of NAD ؉ (transglycosidation). The cellular implications are discussed. A variety of 3-substituted pyridines was found to be substrates for enzyme-catalyzed transglycosidation. A Brö nsted plot of the data yielded a slope of ؉0.98, consistent with the development of a nearly full positive charge in the transition state, and with basicity of the attacking nucleophile as a strong predictor of reactivity. NAD ؉ analogues including ␤-2-deoxy-2-fluororibo-NAD ؉ and a His-to-Ala mutant were used to probe the mechanism of nicotinamide-ribosyl cleavage and acetyl group transfer. We demonstrate that nicotinamide-ribosyl cleavage is distinct from acetyl group transfer to the 2-OH ribose. The observed enzyme-catalyzed formation of a labile 1-acetylated-ADP-fluororibose intermediate using ␤-2-deoxy-2-fluororibo-NAD ؉ supports a mechanism where, after nicotinamide-ribosyl cleavage, the carbonyl oxygen of acetylated substrate attacks the C-1 ribose to form an initial iminium adduct.The acetylation state of histones is intimately coupled to transcription, DNA repair, and replication and is governed by the competing enzymatic activities of histone acetyltransferases and histone deacetylases (reviewed in Refs. 1-3). Recently a new family of histone deacetylases has emerged and is referred to as the silent information regulator 2 (Sir2) 1 family of histone/protein deacetylases (reviewed in Refs. 4 -6) or Sirtuins (7). This family is highly conserved from prokaryotes to humans (7), and there is evidence suggesting that the scope of Sir2 activity extends beyond histone deacetylation and involves other protein targets throughout the cell. In yeast, at least five Sir2-like proteins have been identified. The founding member, yeast Sir2 (ySir2), is required for all major silenced loci (reviewed in Ref. 4). A Sir2 homologue from Salmonella enterica was shown to up-regulate acetyl-CoA synthetase, through deacetylation of a critical lysine residue (8, 9). In humans, seven Sir2 homologues have been identified to date (7). Of these seven, human SIRT2 (hSIRT2) has been identified as a cytosolic protein (10) that deacetylates ␣-tubuli...

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 ADPribose 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.

Sir2 (silent information regulator 2) enzymes catalyze a unique protein deacetylation reaction that requires the coenzyme NAD؉ and produces nicotinamide and a newly discovered metabolite, O-acetyl-ADP-ribose (OAADPr). Conserved from bacteria to humans, these proteins are implicated in the control of gene silencing, metabolism, apoptosis, and aging. Here we examine the role of NAD ؉ metabolites/derivatives and salvage pathway intermediates as activators, inhibitors, or coenzyme substrates of Sir2 enzymes in vitro. Also, we probe the coenzyme binding site using inhibitor binding studies and alternative coenzyme derivatives as substrates. Sir2 enzymes showed an exquisite selectivity for the nicotinamide base coenzyme, with the most dramatic losses in binding affinity/reactivity resulting from relatively minor changes in the nicotinamide ring, either by reduction, as in NADH, or by converting the amide to its acid analogue. Both ends of the dinucleotide NAD ؉ are shown to be critical for high selectivity and high affinity. Among the NAD ؉ metabolites tested none were able to allosterically activate, although all led to various extents of inhibition, consistent with competition at the coenzyme binding site. Nicotinamide was the most potent inhibitor examined, suggesting that cellular nicotinamide levels would provide an effective small molecule regulator of protein deacetylation and generation of OAADPr. The presented findings also suggest that changes in the physiological NAD ؉ :NADH ratio, without a change in NAD ؉ , would yield little alteration in Sir2 activity. That is, NADH is an extremely ineffective inhibitor of Sir2 enzymes (average IC 50 of 17 mM). We propose that changes in both free nicotinamide and free NAD ؉ afford the greatest contribution to cellular activity of Sir2 enzymes but with nicotinamide having a more dramatic effect during smaller fluctuations in concentration.The Sir2 1 (silent information regulator 2) family (or Sirtuins) of protein deacetylases have emerged as important regulators of seemingly diverse cellular processes, such as gene silencing, apoptosis, metabolism, and aging (1-6). These enzymes catalyze a unique reaction that requires the coenzyme NAD ϩ and produces deacetylated product, nicotinamide and O-acetylated ADP-ribose (OAADPr) (7-11). Yeast Sir2 is required for gene silencing at the silent genetic loci and is thought to maintain a hypoacetylated chromatin state by localized histone deacetyation (1). The catalytic core domain is conserved from bacteria to humans, with five homologous genes in yeast and seven in humans (12). Mammalian SIRT1 is localized in the nucleus and has been reported to regulate p53 (reviewed in Ref. 13) and FOXO transcription factors (3, 4), promoting survival under cell stress. Human SIRT2 is cytoplasmic and is reported to deacetylate ␣-tubulin (14), whereas SIRT3 is localized to the matrix of mitochondria (15, 16), although cellular targets have not been reported. Substrates and potential functions of the remaining mammalian homologs have not been demo...