Structural Identification of 2′- and 3′-O-Acetyl-ADP-ribose as Novel Metabolites Derived from the Sir2 Family of β-NAD+-dependent Histone/Protein Deacetylases

The Sir2 (silentinformation regulator 2) family of histone/protein deacetylases has been implicated in a wide range of biological activities, including gene silencing, life-span extension, and chromosomal stability. Their dependence on β-NAD+ for activity is unique among the known classes of histone/protein deacetylase. Sir2 enzymes have been shown to couple substrate deacetylation and β-NAD+ cleavage to the formation of O-acetyl-ADP-ribose, a newly described metabolite. To gain a better understanding of the catalytic mechanism and of the biological implications of producing this molecule, we have performed a detailed enzymatic and structural characterization of O-acetyl-ADP-ribose. Through the use of mass spectrometry, rapid quenching techniques, and NMR structural analyses, 2′-O-acetyl-ADP-ribose and 3′-O-acetyl-ADP-ribose were found to be the solution products produced by the Sir2 family of enzymes. Rapid quenching approaches under single-turnover conditions identified 2′-O-acetyl-ADP-ribose as the enzymatic product, whereas 3′-O-acetyl-ADP-ribose was formed by intramolecular transesterification after enzymatic release into bulk solvent, where 2′- and 3′-O-acetyl-ADP-ribose exist in equilibrium (48:52). In addition to 1H and 13C chemical shift assignments for each regioisomer, heteronuclear multiple-bond correlation spectroscopy was used to assign unambiguously the position of the acetyl group. These findings are highly significant, because they differ from the previous conclusion, which suggested that 1′-O-acetyl-ADP-ribose was the solution product of the reaction. Possible mechanisms for the generation of 2′-O-acetyl-ADP-ribose are discussed.