Structural Basis for the NAD-dependent Deacetylase Mechanism of Sir2

Abstract

The NAD-dependent histone/protein deacetylase activity of Sir2 (silent information regulator 2) accounts for its diverse biological roles including gene silencing, DNA damage repair, cell cycle regulation, and life span extension. We provide crystallographic evidence that 2-O-acetyl ADP-ribose is the reaction product that is formed at the active site of Sir2 from the 2.6-Å co-crystal structure of 2-O-acetyl-ADP-ribose and Sir2 from Archaeoglobus fulgidus. In addition, we show that His-116 and Phe-159 play critical roles in the catalysis and substrate recognition. The conserved Ser-24 and Asp-101 contribute to the stability for NAD binding rather than being directly involved in the catalysis. The crystal structures of wild type and mutant derivatives of Sir2, in conjunction with biochemical analyses of the mutants, provide novel insights into the reaction mechanism of Sir2-mediated deacetylation.

Structural basis for nicotinamide cleavage and ADP-ribose transfer by NAD + -dependent Sir2 histone/protein deacetylases

“…2 a and b). Among the residues that contact the nicotinamide, A33, N116, and D118 are strictly conserved and D43 and F44 are highly conserved among the Sir2 proteins, and mutation of N116 and D118 to alanine abolishes nicotinamide exchange and deacetylation, highlighting the functional importance of the observed contacts (4,10,17). Of particular interest is N116, in which the side-chain carbonyl is bound to a highly ordered water molecule, which would be in position to hydrogen bond to the ring oxygen of the ribose ring of NAD ϩ .…”

“…The acetyllysine and NAD ϩ cosubstrates bind to opposite sides of the cleft and highlight the region of the core domain containing the highest degree of sequence conservation within the Sir2 proteins, implying a conserved catalytic mechanism (3, 7). Biochemical and structural studies reveal that the deacetylation of acetyllysine is coupled to the hydrolysis of NAD ϩ to nicotinamide and 2Ј-O-acetyl-ADP-ribose (8, 9).A detailed structural understanding of how the Sir2 proteins mediate nicotinamide cleavage and ADP-ribose transfer to acetate has been hampered by the difficulty in trapping a Sir2 protein bound to a form of NAD ϩ containing an ordered nicotinamide group (6,7,10,11). To visualize the nicotinamide group of NAD ϩ bound to a Sir2 protein, we now report the high-resolution crystal structure of the Saccharomyces cerevisiae Sir2 homologue, yHst2, bound to an acetyllysine-containing histone H4 peptide and carbanicotinamide adenine dinucleotide (carba-NAD …”

“…A detailed structural understanding of how the Sir2 proteins mediate nicotinamide cleavage and ADP-ribose transfer to acetate has been hampered by the difficulty in trapping a Sir2 protein bound to a form of NAD ϩ containing an ordered nicotinamide group (6,7,10,11). To visualize the nicotinamide group of NAD ϩ bound to a Sir2 protein, we now report the high-resolution crystal structure of the Saccharomyces cerevisiae Sir2 homologue, yHst2, bound to an acetyllysine-containing histone H4 peptide and carbanicotinamide adenine dinucleotide (carba-NAD ϩ ).…”

Ex-527 inhibits Sirtuins by exploiting their unique NAD + -dependent deacetylation mechanism

“…1A), it appears to occupy a conserved pocket (16). Nicotinamide then dissociates from this so-called C-pocket, possibly assisted by further closure of the cofactor binding loop (16,17). The intermediate rearranges to a 1′-2′-bicyclic form, which is finally hydrolyzed to the products 2′-O-acetyl-ADP ribose and deacetylated polypeptide.…”

Identification of Selective Inhibitors of NAD+-dependent Deacetylases Using Phenotypic Screens in Yeast

“…These highly conserved enzymes catalyze a reaction that requires the consumption of NAD ϩ for the removal of the acetyl group from substrate lysine residues to generate nicotinamide, O-acetyl-ADP-ribose, and lysine (3)(4)(5)(6)(7)(8)(9)(10). Initially characterized as histone deacetylases, this family of enzymes was subsequently shown to have a broad range of substrates including p53, BCL6, and ␣-tubulin in mammalian cells and acetyl-CoA synthetase in bacteria (11)(12)(13)(14)(15)(16)(17).…”