Chemistry of Gene Silencing:  The Mechanism of NAD+-Dependent Deacetylation Reactions

• Published in: Biochemistry (Easton) Vol. 40; no. 51; pp. 15456 - 15463
• Main Authors: Sauve, Anthony A, Celic, Ivana, Avalos, Jose, Deng, Haiteng, Boeke, Jef D, Schramm, Vern L
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 25-12-2001
• Subjects: Adenosine Diphosphate Ribose - analogs & derivatives; Adenosine Diphosphate Ribose - chemical synthesis; Adenosine Diphosphate Ribose - chemistry; Adenosine Diphosphate Ribose - metabolism; Amino Acid Sequence; Arabinose - chemistry; Archaeal Proteins - chemistry; Archaeal Proteins - genetics; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Deuterium Oxide - metabolism; Enzyme Inhibitors - chemistry; Fungal Proteins - chemistry; Fungal Proteins - genetics; Gene Silencing; Histone Deacetylase Inhibitors; Histone Deacetylases - chemistry; Histone Deacetylases - genetics; Histone Deacetylases - metabolism; Humans; Isomerism; Kinetics; Molecular Sequence Data; NAD - chemistry; NAD - metabolism; Nuclear Magnetic Resonance, Biomolecular; O-Acetyl-ADP-Ribose; Oxygen Isotopes - metabolism; Silent Information Regulator Proteins, Saccharomyces cerevisiae; Sirtuin 1; Sirtuin 2; Sirtuins; Substrate Specificity; Trans-Activators - antagonists & inhibitors; Trans-Activators - chemistry; Trans-Activators - genetics; Trans-Activators - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi011858j

The Sir2 enzyme family is responsible for a newly classified chemical reaction, NAD+-dependent protein deacetylation. New peptide substrates, the reaction mechanism, and the products of the acetyl transfer to NAD+ are described for SIR2. The final products of SIR2 reactions are the deacetylated peptide and the 2‘ and 3‘ regioisomers of O-acetyl ADP ribose (AADPR), formed through an α-1‘-acetyl ADP ribose intermediate and intramolecular transesterification reactions (2‘ → 3‘). The regioisomers, their anomeric forms, the interconversion rates, and the reaction equilibria were characterized by NMR, HPLC, 18O exchange, and MS methods. The mechanism of acetyl transfer to NAD+ includes (1) ADP ribosylation of the peptide acyl oxygen to form a high-energy O-alkyl amidate intermediate, (2) attack of the 2‘-OH group on the amidate to form a 1‘,2‘-acyloxonium species, (3) hydrolysis to 2‘-AADPR by the attack of water on the carbonyl carbon, and (4) an SIR2-independent transesterification equilibrating the 2‘- and 3‘-AADPRs. This mechanism is unprecedented in ADP-ribosyl transferase enzymology. The 2‘- and 3‘-AADPR products are candidate molecules for SIR2-initiated signaling pathways.