Silent information regulator 2 family of NAD- dependent histone/protein deacetylases generates a unique product, 1- O- acetyl-ADP-ribose

Abstract

Uncovering the true enzymatic function of SIR2 is critical to the basic understanding of its cellular function. Therefore, we set out to authenticate the reaction products and to determine the intrinsic catalytic mechanism. We provide direct evidence that the efficient histone͞protein deacetylase reaction is tightly coupled to the formation of a previously unidentified acetyl-ADP-ribose product (1-O-acetyl-ADP ribose). One molecule of NAD ؉ and one molecule of acetyl-lysine are readily catalyzed to one molecule of deacetylated lysine, nicotinamide, and 1-O-acetyl-ADP-ribose. A unique reaction mechanism involving the attack of enzyme-bound acetate or the direct attack of acetyl-lysine on an oxocarbenium ADP-ribose intermediate is proposed. We suggest that the reported histone͞protein ADPribosyltransferase activity is a low-efficiency side reaction that can be explained through the partial uncoupling of the intrinsic deacetylation and acetate transfer to ADP-ribose.

Sir2 Protein Deacetylases: Evidence for Chemical Intermediates and Functions of a Conserved Histidine

“…In contrast to the primarily nuclear SIRT1, SIRT2 is localized to the cytoplasm where it can deacetylate α-tubulin (23), and SIRT3, SIRT4, and SIRT5 are located in the mitochondrial matrix (24-26), but knowledge of their cellular targets is uncertain. The other human homologs, SIRT6 and SIRT7, are found in the nucleus (24), where SIRT6 is reported to possess ADPribosyltransferase activity (27).The majority of Sir2 homologs are protein deacetylases catalyzing the conversion of NAD + and an acetylated-lysine residue to nicotinamide, the corresponding deacetylated-lysine residue, and 2'-O-acetyl-ADP-ribose (OAADPr) (Scheme 1) (28)(29)(30). This mechanism differs from that of class I and II histone deacetylases (HDACs), in which an active-site zinc directs hydrolysis of the acetylated-lysine residues to acetate and free lysine (31).…”

“…Previously, kinetic analyses have shown that Sir2 deacetylases follow a sequential mechanism in which acetylated protein and then NAD + bind to form a ternary complex before chemical catalysis occurs in two kinetically-distinct steps (37). In the initial chemical step, nicotinamide is released upon formation of an enzyme:ADP-ribose:acetylated protein intermediate, which has been postulated to be an α-1'-O-alkylamidate (Scheme 1) (28)(29)(30). One line of evidence for an α-1'-O-alkylamidate is the ability of nicotinamide, a potent product inhibitor (15,16,(38)(39)(40), to rapidly react with the enzyme:ADP-ribose:acetylated protein intermediate, regenerating NAD + and acetylated protein by a process dubbed the nicotinamide exchange (or transglycosidation) reaction (39,40).…”

Do Sirtuins Promote Mammalian Longevity?: A Critical Review on Its Relevance to the Longevity Effect Induced by Calorie Restriction

“…SIRT1 also deacetylates FOXO1, FOXO3a and FOXO4, modulating cell cycle and reactive oxygen species (ROS) generation by increasing p27kip1, MnSOD (manganese superoxide dismutase), and GADD45 (growth arrest and DNA damage inducible protein 45) (Brunet et al, 2004;Daitoku et al, 2004;Kobayashi et al, 2005;van der Horst et al, 2004). Studies in SIRT1-deficient and overexpressing mice identified SIRT1 as a positive regulator of telomere length, and showed that SIRT1 attenuates telomere shortening associated with aging (Palacios et al, 2010).…”

“…NAD + is hydrolyzed to NAM, which inhibits SIRT1 deacetylase activities (Bitterman et al, 2002;Fulco et al, 2008;Sauve et al, 2005) and O-acetyl-ADP-ribose (Borra et al, 2002;Tanner et al, 2000). Intracellular NAD + levels and SIRT1 function are regulated by nicotinamide phosphoribosyltransferase (NAMPT), which functions to resynthesize NAD + from NAM (Revollo et al, 2004).…”

Potential role of sirtuins in livestock production

“…The nicotinamide-ribosyl bond is broken, and the acetyl group of the substrate is transferred to the ADP-ribose moiety of NAD 1 (Supplementary Figure S1b) Figure S1c; Tanner et al, 2000;Tanny and Moazed, 2001;Marmorstein, 2004;Borra and Denu, 2006).…”