Hydrolysis of O-Acetyl-ADP-ribose Isomers

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 ARH3and ARH1-catalyzed reactions involve nucleophilic attacks at the C-1 position, it was perplexing that theARH3catalytic sitewould cleaveOAADPr at either the 2 or 3 -position, andwe 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 pH9.0 than at neutral pHwhere 3 -OAADPr predominated. Consistent with our hypothesis, IC50 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 H2O resulted in incorporation of one 18O into ADP-ribose by mass spectrometric analysis, consistent with cleavage at the C-1 position. Together, these data suggest that ARH family members, ARH1 andARH3, catalyze hydrolysis of the 1 -O linkage in their structurally diverse substrates.