The silent information regulator 2 (Sir2) family of NAD-dependent N-acetyl-protein deacetylases participates in the regulation of gene silencing, chromatin structure, and longevity. In the Sir2-catalyzed reaction, the acetyl moiety of N-acetyl-histone is transferred to the ADP-ribose of NAD, yielding O-acetyl-ADP-ribose and nicotinamide. We hypothesized that, if O-acetyl-ADP-ribose were an important signaling molecule, a specific hydrolase would cleave the (O-acetyl)-(ADP-ribose) linkage. We report here that the poly-(ADP-ribose) glycohydrolase ARH3 hydrolyzed O-acetyl-ADPribose to produce ADP-ribose in a time-and Mg 2؉ -dependent reaction and thus could participate in two signaling pathways. This O-acetyl-ADP-ribose hydrolase belongs to a family of three structurally related 39-kDa ADP-ribose-binding proteins (ARH1-ARH3). ARH1 was reported to hydrolyze ADP-ribosylarginine, whereas ARH3 degraded poly(ADP-ribose). ARH3-catalyzed generation of ADP-ribose from O-acetyl-ADP-ribose was significantly faster than from poly(ADP-ribose). Like the degradation of poly(ADP-ribose) by ARH3, hydrolysis of O-acetyl-ADP-ribose was abolished by replacement of the vicinal aspartates at positions 77 and 78 of ARH3 with asparagine. The rate of O-acetyl-ADP-ribose hydrolysis by recombinant ARH3 was 250-fold that observed with ARH1; ARH2 and poly(ADP-ribose) glycohydrolase were inactive. All data support the conclusion that the Sir2 reaction product O-acetyl-ADP-ribose is degraded by ARH3.ADP-ribosylhydrolase ͉ ADP-ribosyltransferase ͉ sirtuin ͉ ADP-ribose S ilent information regulator 2 (Sir2) family proteins are involved in gene silencing, chromosomal stability, and lifespan extension (1, 2). In the presence of NAD, Sir2 couples protein deacetylation with formation of O-acetyl-ADP-ribose and release of nicotinamide (3-5). NAD-dependent histone deacetylation appears to be crucial for the biological effects of Sir2. The second product of the reaction, O-acetyl-ADP-ribose, may be involved in the stabilization of chromatin and formation of Sir complexes (6, 7), although its contribution to the biological effects of Sir2 is unclear.In many biological systems, specific enzymes are responsible for the degradation of small molecules that are generated in signaling cascades, thus terminating their action. Examples include the adenylyl cyclase and guanylyl cyclase pathways, where cyclic nucleotide phosphodiesterases degrade cAMP and cGMP, respectively, thus extinguishing the signal (8). Thus far, enzymatic destruction of O-acetyl-ADP-ribose has been shown only with the Nudix family of ADP-ribose pyrophosphatases (9) (nucleoside diphosphate linked to another moiety, hence the acronym Nudix) (10) and perhaps other less selective pyrophosphatases.The extent of ADP-ribosylation of proteins is determined by the rate of opposing actions of ADP-ribosyltransferases, which catalyze the posttranslational modification, and ADP-riboseprotein hydrolases, which cleave the ADP-ribose-protein linkage, releasing ADP-ribose and regenerating unmodified protein...

O-Acetyl-ADP-ribose (OAADPr), produced by the Sir2-catalyzed NAD ؉ -dependent histone/protein deacetylase reaction, regulates diverse biological processes. Interconversion between two OAADPr isomers with acetyl attached to the C-2؆ and C-3؆ hydroxyl of ADP-ribose (ADPr) is rapid. We reported earlier that ADP-ribosylhydrolase 3 (ARH3), one of three ARH proteins sharing structural similarities, hydrolyzed OAADPr to ADPr and acetate, and poly(ADPr) to ADPr monomers. ARH1 also hydrolyzed OAADPr and poly(ADPr) as well as ADP-ribose-arginine, with arginine in ␣-anomeric linkage to C-1؆ of ADP-ribose. Because both ARH3-and ARH1-catalyzed reactions involve nucleophilic attacks at the C-1؆ position, it was perplexing that the ARH3 catalytic site would cleave OAADPr at either the 2؆-or 3؆-position, and we postulated the existence of a third isomer, 1؆-OAADPr, in equilibrium with 2؆-and 3؆-isomers. A third isomer, consistent with 1؆-OAADPr, was identified at pH 9.0. Further, ARH3 OAADPr hydrolase activity was greater at pH 9.0 than at neutral pH where 3؆-OAADPr predominated. Consistent with our hypothesis, IC 50 values for ARH3 inhibition by 2؆-and 3؆-N-acetyl-ADPr analogs of OAADPr were significantly higher than that for ADPr. ARH1 also hydrolyzed OAADPr more rapidly at alkaline pH, but cleavage of ADP-ribose-arginine was faster at neutral pH than pH 9.0. ARH3-catalyzed hydrolysis of OAADPr in H 218 O resulted in incorporation of one 18 O into ADP-ribose by mass spectrometric analysis, consistent with cleavage at the C-1؆ position. Together, these data suggest that ARH family members, ARH1 and ARH3, catalyze hydrolysis of the 1؆-O linkage in their structurally diverse substrates.Mono-ADP-ribosylation is a post-translational modification, in which the ADP-ribose (ADPr) 5 moiety of NAD is transferred to an acceptor protein (1). This modification serves as the mechanism by which several bacterial toxins (e.g. Pseudomonas exoenzyme S, cholera toxin, diphtheria toxin) exert their effects on mammalian cells (2, 3). Mammalian cells also produce endogenous ADP-ribosyltransferases that catalyze reactions similar to the bacterial toxins, specifically, the ADPribosylation of arginine residues in proteins (4). In addition, mammalian cells possess hydrolases that cleave the ADPr-protein linkage, releasing ADPr and regenerating the unmodified protein (5, 6). An ADP-ribosyl(arginine) hydrolase, termed ARH1, catalyzes in a stereospecific manner, hydrolysis of the ␣-linkage of arginine-ribose found in ADP-ribosyl(arginine)-protein to ADPr and (arginine)-protein (7, 8), consistent with the regulation of ADP-ribosyl(arginine)-protein levels by opposing activities of transferases and hydrolases, participating in an ADP-ribosylation cycle (4, 9).Three known members (ARH1-3) of the ARH family of proteins are similar in molecular size (ϳ39 kDa) and amino acid sequence (10). As noted above, ARH1 catalyzes the hydrolysis of ADP-ribose-arginine and also hydrolyzes ADP-ribose linkages to guanidine. The reaction is stereospecific, and only the ␣-ano...

The Sir2 family of NAD‐dependent N‐acetyl‐protein deacetylases participates in the regulation of gene silencing, chromatin structure, and longevity. In the Sir2‐catalyzed reaction, the acetyl moiety of N‐acetyl‐histone is transferred to the ADP‐ribose of NAD, yielding O‐acetyl‐ADP‐ribose and nicotinamide. We hypothesized that if O‐acetyl‐ADP‐ribose were an important signaling molecule, a specific hydrolase would cleave the (O‐acetyl)‐(ADP‐ribose) linkage. We report here that the poly(ADP‐ribose) glycohydrolase ARH3, hydrolyzed O‐acetyl‐ADP‐ribose to produce ADP‐ribose in a time‐ and Mg2+‐dependent reaction and thus could participate in two signaling pathways. This O‐acetyl‐ADP‐ribose hydrolase belongs to a family of three, structurally related 39‐kDa ADP‐ribose‐binding proteins (ARH1‐3). ARH1 was reported to hydrolyze ADP‐ribosylarginine, whereas ARH3 degraded poly(ADP‐ribose). ARH3‐catalyzed generation of ADP‐ribose from O‐acetyl‐ADP‐ribose was significantly faster than from poly(ADP‐ribose). Like the degradation of poly(ADP‐ribose) by ARH3, hydrolysis of O‐acetyl‐ADP‐ribose was abolished by replacement of the vicinal aspartates at positions 77 and 78 of ARH3 with alanine. The rate of O‐acetyl‐ADP‐ribose hydrolysis by recombinant ARH3 was 250‐fold that observed with ARH1; ARH2 and poly‐ADP‐ribose glycohydrolase were inactive. All data support the conclusion that the Sir2 reaction product, O‐acetyl‐ADP‐ribose, is degraded by ARH3.