The Sir2 class (sirtuins) of proteins deacetylases are linked to a wide range of biological functions, that include gene silencing, longevity, survival under stress, fatty acid metabolism, insulin secretion and mitochondrial function. Conserved among all forms of life, most sirtuins display robust NAD+-dependent protein deacetylase activity. Although genetic studies have provided some insight into the pathways controlled by several sirtuins, the molecular basis for the observed biology remains elusive. Our lab has focused on understanding the biochemical basis for sirtuin biology and function. Sirtuins catalyzed the NAD+-dependent deacetylation of target proteins to yield products nicotinamide, deacetylated peptide and the unique metabolite, O-acetyl-ADP-ribose (OAADPr), which has been proposed to act like a second-messenger. We have examined the mechanisms of catalysis, nicotinamide inhibition, ADP-ribosyl transferase activity,and acetyl-peptide specificity. The emerging picture of sirtuin function suggests that the unique nature of this reaction allows these enzymes to tightly couple protein deacetylation and the cellular energy/redox state to the generation of OAADPr, a signaling molecule.