Mechanism of nicotinamide inhibition and transglycosidation by Sir2 histone/protein deacetylases

J Biol Chem. 2003 Dec 19;278(51):50985-98. doi: 10.1074/jbc.M306552200. Epub 2003 Sep 30.


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.

MeSH terms

  • Amino Acid Substitution
  • Catalysis
  • Glycosylation
  • Histone Deacetylases / genetics
  • Histone Deacetylases / physiology*
  • Humans
  • Kinetics
  • Models, Chemical
  • NAD / chemistry
  • Niacinamide / antagonists & inhibitors*
  • Pyridines / chemistry
  • Ribose / chemistry
  • Silent Information Regulator Proteins, Saccharomyces cerevisiae / genetics
  • Silent Information Regulator Proteins, Saccharomyces cerevisiae / physiology
  • Sirtuin 1
  • Sirtuin 2
  • Sirtuins / genetics
  • Sirtuins / physiology*


  • Pyridines
  • Silent Information Regulator Proteins, Saccharomyces cerevisiae
  • NAD
  • Niacinamide
  • Ribose
  • SIR2 protein, S cerevisiae
  • SIRT1 protein, human
  • Sirtuin 1
  • Sirtuin 2
  • Sirtuins
  • Histone Deacetylases