Silent information regulator 2 (Sir2) enzymes catalyze NAD؉ -dependent protein/histone deacetylation, where the acetyl group from the lysine ⑀-amino group is transferred to the ADP-ribose moiety of NAD ؉ , producing nicotinamide and the novel metabolite O-acetyl-ADPribose. Sir2 proteins have been shown to regulate gene silencing, metabolic enzymes, and life span. Recently, nicotinamide has been implicated as a direct negative regulator of cellular Sir2 function; however, the mechanism of nicotinamide inhibition was not established. Sir2 enzymes are multifunctional in that the deacetylase reaction involves the cleavage of the nicotinamide-ribosyl, cleavage of an amide bond, and transfer of the acetyl group ultimately to the 2-ribose hydroxyl of ADP-ribose. Here we demonstrate that nicotinamide inhibition is the result of nicotinamide intercepting an ADP-ribosyl-enzyme-acetyl peptide intermediate with regeneration of NAD ؉ (transglycosidation). The cellular implications are discussed. A variety of 3-substituted pyridines was found to be substrates for enzyme-catalyzed transglycosidation. A Brö nsted plot of the data yielded a slope of ؉0.98, consistent with the development of a nearly full positive charge in the transition state, and with basicity of the attacking nucleophile as a strong predictor of reactivity. NAD ؉ analogues including ␤-2-deoxy-2-fluororibo-NAD ؉ and a His-to-Ala mutant were used to probe the mechanism of nicotinamide-ribosyl cleavage and acetyl group transfer. We demonstrate that nicotinamide-ribosyl cleavage is distinct from acetyl group transfer to the 2-OH ribose. The observed enzyme-catalyzed formation of a labile 1-acetylated-ADP-fluororibose intermediate using ␤-2-deoxy-2-fluororibo-NAD ؉ supports a mechanism where, after nicotinamide-ribosyl cleavage, the carbonyl oxygen of acetylated substrate attacks the C-1 ribose to form an initial iminium adduct.The acetylation state of histones is intimately coupled to transcription, DNA repair, and replication and is governed by the competing enzymatic activities of histone acetyltransferases and histone deacetylases (reviewed in Refs. 1-3). Recently a new family of histone deacetylases has emerged and is referred to as the silent information regulator 2 (Sir2) 1 family of histone/protein deacetylases (reviewed in Refs. 4 -6) or Sirtuins (7). This family is highly conserved from prokaryotes to humans (7), and there is evidence suggesting that the scope of Sir2 activity extends beyond histone deacetylation and involves other protein targets throughout the cell. In yeast, at least five Sir2-like proteins have been identified. The founding member, yeast Sir2 (ySir2), is required for all major silenced loci (reviewed in Ref. 4). A Sir2 homologue from Salmonella enterica was shown to up-regulate acetyl-CoA synthetase, through deacetylation of a critical lysine residue (8, 9). In humans, seven Sir2 homologues have been identified to date (7). Of these seven, human SIRT2 (hSIRT2) has been identified as a cytosolic protein (10) that deacetylates ␣-tubuli...

The Sir2 (silent information 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 ␤-NAD ؉ for activity is unique among the known classes of histone/protein deacetylase. Sir2 enzymes have been shown to couple substrate deacetylation and ␤-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 singleturnover 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 1 H and 13 C 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.The Sir2 (silent information regulator 2) family of enzymes is a unique class of histone/protein deacetylases that are conserved from prokaryotes to humans (1). Investigations into Sir2 function in yeast have revealed that Sir2 is required for silencing at the mating-type loci (2, 3), telomeres (2, 4, 5), and ribosomal DNA (6 -10). Sir2 has also been implicated in lifespan extension within yeast (11-13) and Caenorhabditis elegans (14). Sir2 has been implicated in other cellular processes such as the repair of double-stranded breaks through nonhomologous end joining (15), cell cycle progression, and chromosome stability (16).To date, all Sir2 family of histone/protein deacetylases examined strictly require ␤-NAD ϩ for activity (13,(17)(18)(19). Interestingly, we (17) and others (20) had shown that histone/protein deacetylase activity was tightly coupled to the formation of a previously unidentified product, O-acetyl-ADP-ribose. Initial mass spectral data were consistent with the formation of Oacetyl-ADP-ribose (17). We demonstrated that the deacetylation of substrate and the formation of O-acetyl-ADP-ribose were exquisitely coupled (17). These findings suggested that O-acetyl-ADP-ribose could serve an important function in manifesting the previously reported biological effects of Sir2. In addition to this interesting possibility, it is important to understand how this reactio...

Silent information regulator 2 (Sir2) family of enzymes has been implicated in many cellular processes that include histone deacetylation, gene silencing, chromosomal stability, and aging. Yeast Sir2 and several homologues have been shown to be NAD ؉ -dependent histone/protein deacetylases. Previously, it was demonstrated that the yeast enzymes catalyze a unique reaction mechanism in which the cleavage of NAD ؉ and the deacetylation of substrate are coupled with the formation of O-acetyl-ADP-ribose, a novel metabolite. We demonstrate that the production of O-acetyl-ADP-ribose is evolutionarily conserved among Sir2-like enzymes from yeast, Drosophila, and human. Also, endogenous yeast Sir2 complex from telomeres was shown to generate O-acetyl-ADP-ribose. By using a quantitative microinjection assay to examine the possible biological function(s) of this newly discovered metabolite, we demonstrate that O-acetyl-ADP-ribose causes a delay/block in oocyte maturation and results in a delay/block in embryo cell division in blastomeres. This effect was mimicked by injection of low nanomolar levels of active enzyme but not with a catalytically impaired mutant, indicating that the enzymatic activity is essential for the observed effects. In cell-free oocyte extracts, we demonstrate the existence of cellular enzymes that can efficiently utilize O-acetyl-ADP-ribose.

Introduction: lysine biosynthesis 1.1 The a-aminoadipate pathway to l-lysine 2 Enzymes of the a-aminoadipate pathway 2.1 Homocitrate synthase 2.2 Homoaconitate hydratase 2.3 Homoisocitrate dehydrogenase 2.4 a-Aminoadipate aminotransferase 2.5 a-Aminoadipate reductase 2.6 Saccharopine reductase 2.7 Saccharopine dehydrogenase 3 Role of pipecolic acid in lysine biosynthesis 4 Lysine catabolism 5 Role of a-aminoadipate pathway intermediates in secondary metabolism 6 The a-aminoadipate pathway in archaea and bacteria 7 Conclusion 8 Acknowledgements 9 References

Human PP2Calpha is a metal-dependent phosphoserine/phosphothreonine protein phosphatase and is the representative member of the large PPM family. The X-ray structure of human PP2Calpha has revealed an active site containing a dinuclear metal ion center that is coordinated by several invariant carboxylate residues. However, direct evidence for the catalytic function of these and other active-site residues has not been established. Using site-directed mutagenesis and enzyme kinetic analyses, we probed the roles of conserved active-site amino acids within PP2Calpha. Asp-60 bridges metals M1 and M2, and Asp-239 coordinates metal M2, both of which were replaced individually to asparagine residues. These point mutations resulted in >or=1000-fold decrease in k(cat) and >or=30-fold increase in K(m) value for Mn(2+). Mutation of Asp-282 to asparagine caused a 100-fold decrease in k(cat), but no significant effect on K(m) values for metal and substrate, consistent with Asp-282 activating a metal-bound water nucleophile. Mutants T128A, E37Q, D38N, and H40A displayed little or no alterations on k(cat) and K(m) values for substrate or metal ion (Mn(2+)). Analysis of H62Q and R33A yielded k(cat) values that were 20- and 2-fold lower than wild-type, respectively. The mutant R33A showed a 8-fold higher K(m) for substrate, while the K(m) observed with H62Q was unaffected. A pH-rate profile of the H62Q mutant showed loss of the ionization that must be protonated for activity. Brönsted analysis of substrate leaving group pK(a) values for H62Q indicated a greater dependency (slope -0.84) on leaving group pK(a) in comparison to wild-type (slope -0.33). These data provide strong evidence that His-62 acts as a general acid during the cleavage of the P-O bond.