Structure-based Mechanism of ADP-ribosylation by Sirtuins
2009; Elsevier BV; Volume: 284; Issue: 48 Linguagem: Inglês
10.1074/jbc.m109.024521
ISSN1083-351X
AutoresWilliam F. Hawse, Cynthia Wolberger,
Tópico(s)Calcium signaling and nucleotide metabolism
ResumoSirtuins comprise a family of enzymes found in all organisms, where they play a role in diverse processes including transcriptional silencing, aging, regulation of transcription, and metabolism. The predominant reaction catalyzed by these enzymes is NAD+-dependent lysine deacetylation, although some sirtuins exhibit a weaker ADP-ribosyltransferase activity. Although the Sir2 deacetylation mechanism is well established, much less is known about the Sir2 ADP-ribosylation reaction. We have studied the ADP-ribosylation activity of a bacterial sirtuin, Sir2Tm, and show that acetylated peptides containing arginine or lysine 2 residues C-terminal to the acetyl lysine, the +2 position, are preferentially ADP-ribosylated at the +2 residue. A structure of Sir2Tm bound to the acetylated +2 arginine peptide shows how this arginine could enter the active site and react with a deacetylation reaction intermediate to yield an ADP-ribosylated peptide. The new biochemical and structural studies presented here provide mechanistic insights into the Sir2 ADP-ribosylation reaction and will aid in identifying substrates of this reaction. Sirtuins comprise a family of enzymes found in all organisms, where they play a role in diverse processes including transcriptional silencing, aging, regulation of transcription, and metabolism. The predominant reaction catalyzed by these enzymes is NAD+-dependent lysine deacetylation, although some sirtuins exhibit a weaker ADP-ribosyltransferase activity. Although the Sir2 deacetylation mechanism is well established, much less is known about the Sir2 ADP-ribosylation reaction. We have studied the ADP-ribosylation activity of a bacterial sirtuin, Sir2Tm, and show that acetylated peptides containing arginine or lysine 2 residues C-terminal to the acetyl lysine, the +2 position, are preferentially ADP-ribosylated at the +2 residue. A structure of Sir2Tm bound to the acetylated +2 arginine peptide shows how this arginine could enter the active site and react with a deacetylation reaction intermediate to yield an ADP-ribosylated peptide. The new biochemical and structural studies presented here provide mechanistic insights into the Sir2 ADP-ribosylation reaction and will aid in identifying substrates of this reaction. IntroductionSirtuins, also known as Sir2 enzymes, are a universally conserved class of NAD+-dependent acetyl-lysine specific deacetylases (1.Frye R.A. Biochem. Biophys. Res. Commun. 2000; 273: 793-798Crossref PubMed Scopus (1135) Google Scholar). Sir2 deacetylation substrates include histones (2.Smith J.S. Brachmann C.B. Celic I. Kenna M.A. Muhammad S. Starai V.J. Avalos J.L. Escalante-Semerena J.C. Grubmeyer C. Wolberger C. Boeke J.D. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 6658-6663Crossref PubMed Scopus (612) Google Scholar), FoxO transcription factors (3.Daitoku H. Hatta M. Matsuzaki H. Aratani S. Ohshima T. Miyagishi M. Nakajima T. Fukamizu A. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 10042-10047Crossref PubMed Scopus (505) Google Scholar, 4.Motta M.C. Divecha N. Lemieux M. Kamel C. Chen D. Gu W. Bultsma Y. McBurney M. Guarente L. Cell. 2004; 116: 551-563Abstract Full Text Full Text PDF PubMed Scopus (1179) Google Scholar), p53 (5.Luo J. Nikolaev A.Y. Imai S. Chen D. Su F. Shiloh A. Guarente L. Gu W. Cell. 2001; 107: 137-148Abstract Full Text Full Text PDF PubMed Scopus (1868) Google Scholar), and PGC-1α (6.Rodgers J.T. Lerin C. Haas W. Gygi S.P. Spiegelman B.M. Puigserver P. Nature. 2005; 434: 113-118Crossref PubMed Scopus (2516) Google Scholar, 7.Nemoto S. Fergusson M.M. Finkel T. J. Biol. Chem. 2005; 280: 16456-16460Abstract Full Text Full Text PDF PubMed Scopus (821) Google Scholar). Sir2 enzymes regulate multiple biological pathways including gene silencing (8.Johnson L.M. Kayne P.S. Kahn E.S. Grunstein M. Proc. Natl. Acad. Sci. U.S.A. 1990; 87: 6286-6290Crossref PubMed Scopus (276) Google Scholar, 9.Braunstein M. Rose A.B. Holmes S.G. Allis C.D. Broach J.R. Genes Dev. 1993; 7: 592-604Crossref PubMed Scopus (709) Google Scholar), transcription (3.Daitoku H. Hatta M. Matsuzaki H. Aratani S. Ohshima T. Miyagishi M. Nakajima T. Fukamizu A. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 10042-10047Crossref PubMed Scopus (505) Google Scholar, 4.Motta M.C. Divecha N. Lemieux M. Kamel C. Chen D. Gu W. Bultsma Y. McBurney M. Guarente L. Cell. 2004; 116: 551-563Abstract Full Text Full Text PDF PubMed Scopus (1179) Google Scholar), and fat metabolism (10.Picard F. Kurtev M. Chung N. Topark-Ngarm A. Senawong T. Machado De Oliveira R. Leid M. McBurney M.W. Guarente L. Nature. 2004; 429: 771-776Crossref PubMed Scopus (1632) Google Scholar).The sirtuin deacetylation reaction consumes acetyl lysine and NAD+, yielding deacetylated lysine, nicotinamide, and O-acetyl ADP-ribose (11.Sauve A.A. Celic I. Avalos J. Deng H. Boeke J.D. Schramm V.L. Biochemistry. 2001; 40: 15456-15463Crossref PubMed Scopus (255) Google Scholar). In the first step of the enzymatic reaction, the acetyl lysine reacts with the C1′ of the NAD+ nicotinamide-ribose, leading to the release of nicotinamide and formation of an O-alkylamidate intermediate (11.Sauve A.A. Celic I. Avalos J. Deng H. Boeke J.D. Schramm V.L. Biochemistry. 2001; 40: 15456-15463Crossref PubMed Scopus (255) Google Scholar, 12.Smith B.C. Denu J.M. Biochemistry. 2006; 45: 272-282Crossref PubMed Scopus (90) Google Scholar). Next, the nicotinamide-ribose 2′OH reacts with the O-alkylamidate intermediate to generate a 1′2′-bicyclic species and the deacetylated lysine (11.Sauve A.A. Celic I. Avalos J. Deng H. Boeke J.D. Schramm V.L. Biochemistry. 2001; 40: 15456-15463Crossref PubMed Scopus (255) Google Scholar). Finally, hydrolysis of the 1′2′-bicyclic species yields 2′ O-acetyl ADP-ribose (11.Sauve A.A. Celic I. Avalos J. Deng H. Boeke J.D. Schramm V.L. Biochemistry. 2001; 40: 15456-15463Crossref PubMed Scopus (255) Google Scholar, 13.Jackson M.D. Denu J.M. J. Biol. Chem. 2002; 277: 18535-18544Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). The Sir2 deacetylation reaction is inhibited by the reaction product, nicotinamide(14.Jackson M.D. Schmidt M.T. Oppenheimer N.J. Denu J.M. J. Biol. Chem. 2003; 278: 50985-50998Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 15.Sauve A.A. Schramm V.L. Biochemistry. 2003; 42: 9249-9256Crossref PubMed Scopus (195) Google Scholar), which binds to the Sir2 active site and reacts with the O-alkylamidate to regenerate NAD+ and acetyl lysine.Alhough Sir2 enzymes are known primarily as protein deacetylases, sirtuins were first identified as NAD+-dependent ADP-ribosyltransferases (16.Tanny J.C. Dowd G.J. Huang J. Hilz H. Moazed D. Cell. 1999; 99: 735-745Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar). The Escherichia coli sirtuin, CobB, ribosylates a small molecule, 5,6-dimethylbenzimidazole (17.Tsang A.W. Escalante-Semerena J.C. J. Biol. Chem. 1998; 273: 31788-31794Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 18.Frye R.A. Biochem. Biophys. Res. Commun. 1999; 260: 273-279Crossref PubMed Scopus (652) Google Scholar). Subsequent studies demonstrated that CobB can also ADP-ribosylate protein substrates (18.Frye R.A. Biochem. Biophys. Res. Commun. 1999; 260: 273-279Crossref PubMed Scopus (652) Google Scholar). Other sirtuins including human SIRT1(19.Liszt G. Ford E. Kurtev M. Guarente L. J. Biol. Chem. 2005; 280: 21313-21320Abstract Full Text Full Text PDF PubMed Scopus (441) Google Scholar), human SIRT4 (20.Haigis M.C. Mostoslavsky R. Haigis K.M. Fahie K. Christodoulou D.C. Murphy A.J. Valenzuela D.M. Yancopoulos G.D. Karow M. Blander G. Wolberger C. Prolla T.A. Weindruch R. Alt F.W. Guarente L. Cell. 2006; 126: 941-954Abstract Full Text Full Text PDF PubMed Scopus (927) Google Scholar), and a trypanosomal sirtuin (21.García-Salcedo J.A. Gijón P. Nolan D.P. Tebabi P. Pays E. EMBO J. 2003; 22: 5851-5862Crossref PubMed Scopus (110) Google Scholar) can ADP-ribosylate protein substrates. The Trypanosoma brucei Sir2 enzyme, TbSir2Rp1, is a particularly well characterized sirtuin with ADP-ribosyltransferase activity. TbSir2Rp1 has dual deacetylase and ADP-ribosyltransferase activities on histone substrates (21.García-Salcedo J.A. Gijón P. Nolan D.P. Tebabi P. Pays E. EMBO J. 2003; 22: 5851-5862Crossref PubMed Scopus (110) Google Scholar); however, its deacetylation activity is 5 orders of magnitude greater than its ADP-ribosyltransferase activity (22.Kowieski T.M. Lee S. Denu J.M. J. Biol. Chem. 2008; 283: 5317-5326Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The TbSir2Rp1 ribosylation activity is greatly enhanced by acetyl lysine (22.Kowieski T.M. Lee S. Denu J.M. J. Biol. Chem. 2008; 283: 5317-5326Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). One model that could explain the increased Sir2 ADP-ribosyltransferase activity with acetylated substrates is that a deacetylation reaction intermediate, possibly the O-alkylamidate intermediate, reacts with a nucleophilic amino acid on the substrate protein to yield an ADP-ribosylated product (22.Kowieski T.M. Lee S. Denu J.M. J. Biol. Chem. 2008; 283: 5317-5326Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). How a nucleophilic side chain enters the Sir2 active site or what amino acid side chains can be ADP-ribosylated by sirtuins is not known.In structures of sirtuins bound to an acetylated p53 peptide and to an S-alkylamidate intermediate (23.Hawse W.F. Hoff K.G. Fatkins D.G. Daines A. Zubkova O.V. Schramm V.L. Zheng W. Wolberger C. Structure. 2008; 16: 1368-1377Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 24.Cosgrove M.S. Bever K. Avalos J.L. Muhammad S. Zhang X. Wolberger C. Biochemistry. 2006; 45: 7511-7521Crossref PubMed Scopus (82) Google Scholar, 25.Avalos J.L. Celic I. Muhammad S. Cosgrove M.S. Boeke J.D. Wolberger C. Mol Cell. 2002; 10: 523-535Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 26.Hoff K.G. Avalos J.L. Sens K. Wolberger C. Structure. 2006; 14: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 27.Avalos J.L. Boeke J.D. Wolberger C. Mol Cell. 2004; 13: 639-648Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar), a methionine located two residues C-terminal to the acetyl lysine, the +2 position, inserts into the Sir2 active site (see Fig. 1A). This suggested that substrates containing nucleophilic side chains at this position could potentially attack the O-alkylamidate intermediate and become ADP-ribosylated. To test this model, we performed a series of biochemical assays on the Thermotoga maritima sirtuin, Sir2Tm, and the mammalian sirtuin, SIRT1, using various peptides and found that these sirtuins ADP-ribosylate arginine at the +2 position in an acetylated peptide substrate. To gain further mechanistic insights, we solved a structure of Sir2Tm bound to the Arg-acetylated +2 peptide. The structural and biochemical data presented here describe a plausible mechanism for Sir2-mediated ADP-ribosylation of acetylated substrates.DISCUSSIONThe work presented here provides mechanistic insights into how acetylated substrates exhibit enhanced ADP-ribosylation by Sir2Tm. In several structures of either Sir2Tm or Sir2Af2 bound to an acetylated p53 peptide, the peptide methionine located two amino acids C-terminal to the acetyl lysine inserts into the enzyme active site just adjacent to the acetyl lysine (23.Hawse W.F. Hoff K.G. Fatkins D.G. Daines A. Zubkova O.V. Schramm V.L. Zheng W. Wolberger C. Structure. 2008; 16: 1368-1377Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 24.Cosgrove M.S. Bever K. Avalos J.L. Muhammad S. Zhang X. Wolberger C. Biochemistry. 2006; 45: 7511-7521Crossref PubMed Scopus (82) Google Scholar, 25.Avalos J.L. Celic I. Muhammad S. Cosgrove M.S. Boeke J.D. Wolberger C. Mol Cell. 2002; 10: 523-535Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 26.Hoff K.G. Avalos J.L. Sens K. Wolberger C. Structure. 2006; 14: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Previously, Kowieski et al. (22.Kowieski T.M. Lee S. Denu J.M. J. Biol. Chem. 2008; 283: 5317-5326Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) proposed that a nucleophilic side chain on an acetylated protein substrate could intercept a Sir2 deacetylation reaction intermediate. This proposal and previous Sir2-acetylated peptide structures led us to speculate that a nucleophilic amino acid substituted at the +2 position could react with the O-alkylamidate intermediate to yield an ADP-ribosylated product. Based on the dimensions and geometry of the Sir2Tm active site (26.Hoff K.G. Avalos J.L. Sens K. Wolberger C. Structure. 2006; 14: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), the +2 amino acid from the substrate peptide needs to be longer than 5 Å to enter the active site, and ideally, longer than 6 Å. We found that substituting lysine or arginine at this key position of the acetylated p53 peptide indeed results in increased enzymatic ADP-ribosylation by Sir2Tm (Figs. 1, B and D, and 2). The acetyl p53 +2 Arg peptide also stimulates ADP-ribosylation activity of SIRT1 (Fig. 1E), suggesting that ADP-ribosylation activity is conserved in at least a subset of Sir2 enzymes. Peptides with arginine substitutions at positions other than two amino acids C-terminal to the acetyl lysine are far less efficiently ADP-ribosylated (Fig. 2), indicating that ADP-ribosylation by Sir2Tm is sensitive to the position of the substituted arginine. Acetyl lysine residues that are substrates for Sir2 enzymes are located in loop regions of proteins, so using peptide substrates is reasonable. However, it would be informative to analyze the Sir2 ribosylation activity on a protein substrate.The +2 Arg of the acetylated peptide was directly ADP-ribosylated by Sir2Tm. Based on the structure of Sir2Tm bound to the acetyl p53 +2 Arg peptide, the +2 Arg enters the Sir2Tm active site adjacent to the acetyl lysine binding tunnel. In this position, the +2 arginine could react with a Sir2 deacetylation reaction intermediate to become ADP-ribosylated. Our biochemical and structural data support a mechanism in which the +2 arginine reacts with the O-alkylamidate intermediate generating ADP-ribosylated arginine, nicotinamide, and acetyl lysine (Fig. 6, step I). Next, the +2 arginine would react with the O-alkylamidate to generate ADP-ribosylated arginine, nicotinamide, and acetyl lysine (Fig. 6, step II).FIGURE 6Structure-based mechanism of acetyl-dependent Sir2Tm-mediated ribosylation. In the first step of the reaction (step I), the acetyl lysine reacts with NAD+ to generate the O-alkylamidate. The O-alkylamidate can react with nicotinamide to regenerate the starting reactants, NAD+ and acetyl lysine, react with the 2′OH from the nicotinamide-ribose, leading to deacetylation products, or react with the arginine, resulting in ribosylation of the peptide substrate (step II).View Large Image Figure ViewerDownload Hi-res image Download (PPT)An N-terminal lysine is also ADP-ribosylated in the acetylated +2 Arg peptide, suggesting an alternate pathway for ADP-ribosylating substrates (Fig. 3). In structures of Sir2Tm bound to the acetylated p53 peptide, this N-terminal lysine cannot access the Sir2Tm active site. The N-terminal lysine is disordered in all of the Sir2Tm-acetyl p53 peptide complex structures and extends out into solvent. There are at least three models that could explain the ADP-ribosylation of this lysine. First, the reaction product O-acetyl ADP-ribose or NAD+ could react with this lysine nonenzymatically to yield a ribosylated product. There are three additional lysines in the acetyl p53 peptide that are not ribosylated (Fig. 2), arguing that this nonenzymatic mechanism is not likely. A second model would involve binding of a first acetyl peptide and formation of the O-alkylamidate intermediate. Next, another peptide could bind to Sir2Tm and insert the N-terminal lysine into the active site to react with the O-alkylamidate intermediate or another deacetylation intermediate. Based on all identified entry tunnels to the Sir2 active site, the lysine from the second peptide would not be long enough to gain access to the active site, suggesting that this mechanism is not likely. A third model involves Sir2Tm binding the N-terminal lysine through the acetyl lysine binding tunnel instead of the acetyl lysine. A crystal structure of Sir2Tm bound to an unacetylated p53 peptide demonstrates that Sir2Tm can bind unacetylated lysine peptides within the acetyl lysine binding tunnel (24.Cosgrove M.S. Bever K. Avalos J.L. Muhammad S. Zhang X. Wolberger C. Biochemistry. 2006; 45: 7511-7521Crossref PubMed Scopus (82) Google Scholar). The unacetylated lysine can insert into the active site and come within 4.5 Å of the C1′ of NAD+ (Fig. 7). Next, the lysine could then directly attack the C1′ of NAD+ to yield a ribosylated lysine and nicotinamide. Further studies will be required to assess this proposed mechanism and determine whether other post-translational modifications including propionyl-lysine, which is a substrate for Sir2Tm-mediated depropionylation(33.Garrity J. Gardner J.G. Hawse W. Wolberger C. Escalante-Semerena J.C. J. Biol. Chem. 2007; 282: 30239-30245Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar), are substrates for the Sir2 ribosylation reaction.FIGURE 7Model of Sir2Tm bound to deacetylated peptide and NAD+. A model based on the Sir2Tm deacetylated peptide of Sir2Tm bound to deacetylated p53 peptide and NAD+ was built using the Quanta software package (Accelerys). The peptide lysine and NAD+ are colored in yellow, and the conserved histidine 116 is colored in gray.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The significance of the ADP-ribosylation activity of sirtuins to their physiological function remains an open question. The best established role for this activity is the regulation of the mitochondrial enzyme, glutamate dehydrogenase, by SIRT4 (20.Haigis M.C. Mostoslavsky R. Haigis K.M. Fahie K. Christodoulou D.C. Murphy A.J. Valenzuela D.M. Yancopoulos G.D. Karow M. Blander G. Wolberger C. Prolla T.A. Weindruch R. Alt F.W. Guarente L. Cell. 2006; 126: 941-954Abstract Full Text Full Text PDF PubMed Scopus (927) Google Scholar). Glutamate dehydrogenase had previously been shown to be inactivated by ADP-ribosylation, and SIRT4 was subsequently identified as the enzyme responsible for the ADP-ribosylation (20.Haigis M.C. Mostoslavsky R. Haigis K.M. Fahie K. Christodoulou D.C. Murphy A.J. Valenzuela D.M. Yancopoulos G.D. Karow M. Blander G. Wolberger C. Prolla T.A. Weindruch R. Alt F.W. Guarente L. Cell. 2006; 126: 941-954Abstract Full Text Full Text PDF PubMed Scopus (927) Google Scholar). Although it is not known whether ADP-ribosylation of glutamate dehydrogenase is dependent upon prior acetylation of that enzyme, it was recently found that glutamate dehydrogenase is acetylated at multiple sites (34.Choudhary C. Kumar C. Gnad F. Nielsen M.L. Rehman M. Walther T.C. Olsen J.V. Mann M. Science. 2009; 325: 834-840Crossref PubMed Scopus (3079) Google Scholar), raising the possibility that ADP-ribosylation of this enzyme could proceed as described here for Sir2Tm. Although other mono-ADP-ribosylated proteins have been identified in eukaryotic cells (35.Leno G.H. Ledford B.E. FEBS Lett. 1990; 276: 29-33Crossref PubMed Scopus (41) Google Scholar), it remains to be determined which enzymes are responsible for adding this post-translational modification in vivo. IntroductionSirtuins, also known as Sir2 enzymes, are a universally conserved class of NAD+-dependent acetyl-lysine specific deacetylases (1.Frye R.A. Biochem. Biophys. Res. Commun. 2000; 273: 793-798Crossref PubMed Scopus (1135) Google Scholar). Sir2 deacetylation substrates include histones (2.Smith J.S. Brachmann C.B. Celic I. Kenna M.A. Muhammad S. Starai V.J. Avalos J.L. Escalante-Semerena J.C. Grubmeyer C. Wolberger C. Boeke J.D. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 6658-6663Crossref PubMed Scopus (612) Google Scholar), FoxO transcription factors (3.Daitoku H. Hatta M. Matsuzaki H. Aratani S. Ohshima T. Miyagishi M. Nakajima T. Fukamizu A. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 10042-10047Crossref PubMed Scopus (505) Google Scholar, 4.Motta M.C. Divecha N. Lemieux M. Kamel C. Chen D. Gu W. Bultsma Y. McBurney M. Guarente L. Cell. 2004; 116: 551-563Abstract Full Text Full Text PDF PubMed Scopus (1179) Google Scholar), p53 (5.Luo J. Nikolaev A.Y. Imai S. Chen D. Su F. Shiloh A. Guarente L. Gu W. Cell. 2001; 107: 137-148Abstract Full Text Full Text PDF PubMed Scopus (1868) Google Scholar), and PGC-1α (6.Rodgers J.T. Lerin C. Haas W. Gygi S.P. Spiegelman B.M. Puigserver P. Nature. 2005; 434: 113-118Crossref PubMed Scopus (2516) Google Scholar, 7.Nemoto S. Fergusson M.M. Finkel T. J. Biol. Chem. 2005; 280: 16456-16460Abstract Full Text Full Text PDF PubMed Scopus (821) Google Scholar). Sir2 enzymes regulate multiple biological pathways including gene silencing (8.Johnson L.M. Kayne P.S. Kahn E.S. Grunstein M. Proc. Natl. Acad. Sci. U.S.A. 1990; 87: 6286-6290Crossref PubMed Scopus (276) Google Scholar, 9.Braunstein M. Rose A.B. Holmes S.G. Allis C.D. Broach J.R. Genes Dev. 1993; 7: 592-604Crossref PubMed Scopus (709) Google Scholar), transcription (3.Daitoku H. Hatta M. Matsuzaki H. Aratani S. Ohshima T. Miyagishi M. Nakajima T. Fukamizu A. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 10042-10047Crossref PubMed Scopus (505) Google Scholar, 4.Motta M.C. Divecha N. Lemieux M. Kamel C. Chen D. Gu W. Bultsma Y. McBurney M. Guarente L. Cell. 2004; 116: 551-563Abstract Full Text Full Text PDF PubMed Scopus (1179) Google Scholar), and fat metabolism (10.Picard F. Kurtev M. Chung N. Topark-Ngarm A. Senawong T. Machado De Oliveira R. Leid M. McBurney M.W. Guarente L. Nature. 2004; 429: 771-776Crossref PubMed Scopus (1632) Google Scholar).The sirtuin deacetylation reaction consumes acetyl lysine and NAD+, yielding deacetylated lysine, nicotinamide, and O-acetyl ADP-ribose (11.Sauve A.A. Celic I. Avalos J. Deng H. Boeke J.D. Schramm V.L. Biochemistry. 2001; 40: 15456-15463Crossref PubMed Scopus (255) Google Scholar). In the first step of the enzymatic reaction, the acetyl lysine reacts with the C1′ of the NAD+ nicotinamide-ribose, leading to the release of nicotinamide and formation of an O-alkylamidate intermediate (11.Sauve A.A. Celic I. Avalos J. Deng H. Boeke J.D. Schramm V.L. Biochemistry. 2001; 40: 15456-15463Crossref PubMed Scopus (255) Google Scholar, 12.Smith B.C. Denu J.M. Biochemistry. 2006; 45: 272-282Crossref PubMed Scopus (90) Google Scholar). Next, the nicotinamide-ribose 2′OH reacts with the O-alkylamidate intermediate to generate a 1′2′-bicyclic species and the deacetylated lysine (11.Sauve A.A. Celic I. Avalos J. Deng H. Boeke J.D. Schramm V.L. Biochemistry. 2001; 40: 15456-15463Crossref PubMed Scopus (255) Google Scholar). Finally, hydrolysis of the 1′2′-bicyclic species yields 2′ O-acetyl ADP-ribose (11.Sauve A.A. Celic I. Avalos J. Deng H. Boeke J.D. Schramm V.L. Biochemistry. 2001; 40: 15456-15463Crossref PubMed Scopus (255) Google Scholar, 13.Jackson M.D. Denu J.M. J. Biol. Chem. 2002; 277: 18535-18544Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). The Sir2 deacetylation reaction is inhibited by the reaction product, nicotinamide(14.Jackson M.D. Schmidt M.T. Oppenheimer N.J. Denu J.M. J. Biol. Chem. 2003; 278: 50985-50998Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 15.Sauve A.A. Schramm V.L. Biochemistry. 2003; 42: 9249-9256Crossref PubMed Scopus (195) Google Scholar), which binds to the Sir2 active site and reacts with the O-alkylamidate to regenerate NAD+ and acetyl lysine.Alhough Sir2 enzymes are known primarily as protein deacetylases, sirtuins were first identified as NAD+-dependent ADP-ribosyltransferases (16.Tanny J.C. Dowd G.J. Huang J. Hilz H. Moazed D. Cell. 1999; 99: 735-745Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar). The Escherichia coli sirtuin, CobB, ribosylates a small molecule, 5,6-dimethylbenzimidazole (17.Tsang A.W. Escalante-Semerena J.C. J. Biol. Chem. 1998; 273: 31788-31794Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 18.Frye R.A. Biochem. Biophys. Res. Commun. 1999; 260: 273-279Crossref PubMed Scopus (652) Google Scholar). Subsequent studies demonstrated that CobB can also ADP-ribosylate protein substrates (18.Frye R.A. Biochem. Biophys. Res. Commun. 1999; 260: 273-279Crossref PubMed Scopus (652) Google Scholar). Other sirtuins including human SIRT1(19.Liszt G. Ford E. Kurtev M. Guarente L. J. Biol. Chem. 2005; 280: 21313-21320Abstract Full Text Full Text PDF PubMed Scopus (441) Google Scholar), human SIRT4 (20.Haigis M.C. Mostoslavsky R. Haigis K.M. Fahie K. Christodoulou D.C. Murphy A.J. Valenzuela D.M. Yancopoulos G.D. Karow M. Blander G. Wolberger C. Prolla T.A. Weindruch R. Alt F.W. Guarente L. Cell. 2006; 126: 941-954Abstract Full Text Full Text PDF PubMed Scopus (927) Google Scholar), and a trypanosomal sirtuin (21.García-Salcedo J.A. Gijón P. Nolan D.P. Tebabi P. Pays E. EMBO J. 2003; 22: 5851-5862Crossref PubMed Scopus (110) Google Scholar) can ADP-ribosylate protein substrates. The Trypanosoma brucei Sir2 enzyme, TbSir2Rp1, is a particularly well characterized sirtuin with ADP-ribosyltransferase activity. TbSir2Rp1 has dual deacetylase and ADP-ribosyltransferase activities on histone substrates (21.García-Salcedo J.A. Gijón P. Nolan D.P. Tebabi P. Pays E. EMBO J. 2003; 22: 5851-5862Crossref PubMed Scopus (110) Google Scholar); however, its deacetylation activity is 5 orders of magnitude greater than its ADP-ribosyltransferase activity (22.Kowieski T.M. Lee S. Denu J.M. J. Biol. Chem. 2008; 283: 5317-5326Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The TbSir2Rp1 ribosylation activity is greatly enhanced by acetyl lysine (22.Kowieski T.M. Lee S. Denu J.M. J. Biol. Chem. 2008; 283: 5317-5326Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). One model that could explain the increased Sir2 ADP-ribosyltransferase activity with acetylated substrates is that a deacetylation reaction intermediate, possibly the O-alkylamidate intermediate, reacts with a nucleophilic amino acid on the substrate protein to yield an ADP-ribosylated product (22.Kowieski T.M. Lee S. Denu J.M. J. Biol. Chem. 2008; 283: 5317-5326Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). How a nucleophilic side chain enters the Sir2 active site or what amino acid side chains can be ADP-ribosylated by sirtuins is not known.In structures of sirtuins bound to an acetylated p53 peptide and to an S-alkylamidate intermediate (23.Hawse W.F. Hoff K.G. Fatkins D.G. Daines A. Zubkova O.V. Schramm V.L. Zheng W. Wolberger C. Structure. 2008; 16: 1368-1377Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 24.Cosgrove M.S. Bever K. Avalos J.L. Muhammad S. Zhang X. Wolberger C. Biochemistry. 2006; 45: 7511-7521Crossref PubMed Scopus (82) Google Scholar, 25.Avalos J.L. Celic I. Muhammad S. Cosgrove M.S. Boeke J.D. Wolberger C. Mol Cell. 2002; 10: 523-535Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 26.Hoff K.G. Avalos J.L. Sens K. Wolberger C. Structure. 2006; 14: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 27.Avalos J.L. Boeke J.D. Wolberger C. Mol Cell. 2004; 13: 639-648Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar), a methionine located two residues C-terminal to the acetyl lysine, the +2 position, inserts into the Sir2 active site (see Fig. 1A). This suggested that substrates containing nucleophilic side chains at this position could potentially attack the O-alkylamidate intermediate and become ADP-ribosylated. To test this model, we performed a series of biochemical assays on the Thermotoga maritima sirtuin, Sir2Tm, and the mammalian sirtuin, SIRT1, using various peptides and found that these sirtuins ADP-ribosylate arginine at the +2 position in an acetylated peptide substrate. To gain further mechanistic insights, we solved a structure of Sir2Tm bound to the Arg-acetylated +2 peptide. The structural and biochemical data presented here describe a plausible mechanism for Sir2-mediated ADP-ribosylation of acetylated substrates.
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