title: Mechanism of nicotinamide inhibition and transglycosidation by Sir2 histone/protein deacetylases.
abstract: Silent information regulator 2 (Sir2) enzymes catalyze NAD+-dependent protein/histone deacetylation, where the acetyl group from the lysine epsilon-amino group is transferred to the ADP-ribose moiety of NAD+, producing nicotinamide and the novel metabolite O-acetyl-ADP-ribose. 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 Brönsted 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 beta-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 beta-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.
authors.affiliation: Oregon Health and Sciences University, Department of Biochemistry and Molecular Biology, Portland, Oregon 97201-3089, USA.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Jackson
authors.forename: Manning T
authors.initials: MT
authors.lastname: Schmidt
authors.forename: Norman J
authors.initials: NJ
authors.lastname: Oppenheimer
authors.forename: John M
authors.initials: JM
authors.lastname: Denu
mesh_terms: D019943:Amino Acid Substitution; D002384:Catalysis; D006031:Glycosylation; D006655:Histone Deacetylases / Q000235:genetics / Q000502:physiology*; D006801:Humans; D007700:Kinetics; D008956:Models, Chemical; D009243:NAD / Q000737:chemistry; D009536:Niacinamide / Q000037:antagonists & inhibitors*; D011725:Pyridines / Q000737:chemistry; D012266:Ribose / Q000737:chemistry; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae / Q000235:genetics / Q000502:physiology; D056564:Sirtuin 1; D056565:Sirtuin 2; D037761:Sirtuins / Q000235:genetics / Q000502:physiology*
chemical_list: D011725:Pyridines; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae; D009243:NAD; D009536:Niacinamide; D012266:Ribose; C087778:SIR2 protein, S cerevisiae; C447939:SIRT1 protein, human; D056564:Sirtuin 1; D056565:Sirtuin 2; D037761:Sirtuins; D006655:Histone Deacetylases