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

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- 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- ribosylenzyme- 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 Bronsted 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.