ABSTRACT
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-2â³ and C-3â³ 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 α-anomeric linkage to C-1â³ of ADP-ribose. Because both ARH3- and ARH1-catalyzed reactions involve nucleophilic attacks at the C-1â³ position, it was perplexing that the ARH3 catalytic site would cleave OAADPr at either the 2â³- or 3â³-position, and we postulated the existence of a third isomer, 1â³-OAADPr, in equilibrium with 2â³- and 3â³-isomers. A third isomer, consistent with 1â³-OAADPr, was identified at pH 9.0. Further, ARH3 OAADPr hydrolase activity was greater at pH 9.0 than at neutral pH where 3â³-OAADPr predominated. Consistent with our hypothesis, IC(50) values for ARH3 inhibition by 2â³- and 3â³-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(2)(18)O resulted in incorporation of one (18)O into ADP-ribose by mass spectrometric analysis, consistent with cleavage at the C-1â³ position. Together, these data suggest that ARH family members, ARH1 and ARH3, catalyze hydrolysis of the 1â³-O linkage in their structurally diverse substrates.
Subject(s)
Glycoside Hydrolases/chemistry , N-Glycosyl Hydrolases/chemistry , O-Acetyl-ADP-Ribose/metabolism , Adenosine Diphosphate Ribose/chemistry , Catalysis , Catalytic Domain , Gene Expression Regulation, Enzymologic , Hydrogen-Ion Concentration , Hydrolysis , Inhibitory Concentration 50 , Models, Chemical , Models, Theoretical , Poly Adenosine Diphosphate Ribose/chemistry , Protein Isoforms , Sirtuin 1/chemistry , Sirtuins/chemistryABSTRACT
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-ADP-ribose 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.
