title: Structural identification of 2'- and 3'-O-acetyl-ADP-ribose as novel metabolites derived from the Sir2 family of beta -NAD+-dependent histone/protein deacetylases.
abstract: The Sir2 (silent information regulator 2) family of histone/protein deacetylases has been implicated in a wide range of biological activities, including gene silencing, life-span extension, and chromosomal stability. Their dependence on beta-NAD(+) for activity is unique among the known classes of histone/protein deacetylase. Sir2 enzymes have been shown to couple substrate deacetylation and beta-NAD(+) cleavage to the formation of O-acetyl-ADP-ribose, a newly described metabolite. To gain a better understanding of the catalytic mechanism and of the biological implications of producing this molecule, we have performed a detailed enzymatic and structural characterization of O-acetyl-ADP-ribose. Through the use of mass spectrometry, rapid quenching techniques, and NMR structural analyses, 2'-O-acetyl-ADP-ribose and 3'-O-acetyl-ADP-ribose were found to be the solution products produced by the Sir2 family of enzymes. Rapid quenching approaches under single-turnover conditions identified 2'-O-acetyl-ADP-ribose as the enzymatic product, whereas 3'-O-acetyl-ADP-ribose was formed by intramolecular transesterification after enzymatic release into bulk solvent, where 2'- and 3'-O-acetyl-ADP-ribose exist in equilibrium (48:52). In addition to (1)H and (13)C chemical shift assignments for each regioisomer, heteronuclear multiple-bond correlation spectroscopy was used to assign unambiguously the position of the acetyl group. These findings are highly significant, because they differ from the previous conclusion, which suggested that 1'-O-acetyl-ADP-ribose was the solution product of the reaction. Possible mechanisms for the generation of 2'-O-acetyl-ADP-ribose are discussed.
authors.affiliation: Oregon Health & Science University, Department of Biochemistry and Molecular Biology, Portland, Oregopn 97201-3098, USA.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Jackson
authors.forename: John M
authors.initials: JM
authors.lastname: Denu
mesh_terms: D000246:Adenosine Diphosphate Ribose / Q000031:analogs & derivatives / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D006655:Histone Deacetylases / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009243:NAD / Q000378:metabolism*; D038961:O-Acetyl-ADP-Ribose; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae; D056565:Sirtuin 2; D037761:Sirtuins; D015534:Trans-Activators / Q000378:metabolism*
chemical_list: D038961:O-Acetyl-ADP-Ribose; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae; D015534:Trans-Activators; D009243:NAD; D000246:Adenosine Diphosphate Ribose; C087778:SIR2 protein, S cerevisiae; D056565:Sirtuin 2; D037761:Sirtuins; D006655:Histone Deacetylases