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. 2008 Sep 10;16(9):1368-77.
doi: 10.1016/j.str.2008.05.015.

Structural insights into intermediate steps in the Sir2 deacetylation reaction

William F Hawse  1 , Kevin G Hoff, David G Fatkins, Alison Daines, Olga V Zubkova, Vern L Schramm, Weiping Zheng, Cynthia Wolberger
Affiliations

Structural insights into intermediate steps in the Sir2 deacetylation reaction

William F Hawse et al. Structure. .

Abstract

Sirtuin enzymes comprise a unique class of NAD(+)-dependent protein deacetylases. Although structures of many sirtuin complexes have been determined, structural resolution of intermediate chemical steps are needed to understand the deacetylation mechanism. We report crystal structures of the bacterial sirtuin, Sir2Tm, in complex with an S-alkylamidate intermediate, analogous to the naturally occurring O-alkylamidate intermediate, and a Sir2Tm ternary complex containing a dissociated NAD(+) analog and acetylated peptide. The structures and biochemical studies reveal critical roles for the invariant active site histidine in positioning the reaction intermediate, and for a conserved phenylalanine residue in shielding reaction intermediates from base exchange with nicotinamide. The new structural and biochemical studies provide key mechanistic insight into intermediate steps of the Sir2 deacetylation reaction.

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Figures

Figure 1
Figure 1. Sirtuin deacetylation reaction mechanism
(A) In the first step of the Sir2 Deacetylation reaction (I), the ADP ribose moiety of NAD+ is transferred to acetyl lysine generating the O-Alkylamidate intermediate. In this step (I), the Nicotinamide-N-ribose bond is broken to generate free nicotinamide. Next, the N-ribose 2’OH group attacks the O-alkylamidate intermediate generating a 1’ 2’ bicyclic species (II). Subsequent hydrolysis of the 1’ 2’ bicyclic species yields deacetylated lysine and 2’ O-acetyl ADP ribose (III). The structure of the DADMe-NAD+ analogue, which represents a dissociated NAD+ species, is depicted in panel (B).
Figure 2
Figure 2. Structure of a Sir2TM-acetylated peptide – DADMe-NAD+ analogue ternary complex
(A) The 2Fo-Fc (1σ) electron density maps defines the position of the acetyl group and DADMe-NAD+ analogue. (B) Overlaying the Sir2-acetyl peptide DADMe-NAD+ structure (colored in blue) with the Sir2 Michaelis complex (colored in yellow) reveals that the nicotinamide and acetyl lysine moieties remain fixed, while the N-ribose moiety of the DADMe-NAD+ molecule is closer to acetyl lysine.
Figure 3
Figure 3. Structure of a Sir2-S-Alkylamidate intermediate complex
The Fo-Fc omit electron density map (2.5σ) and 2Fo-Fc (1σ) electron density map of the solved complex defining the S-alkylamidate intermediate are displayed in panels (A) and (B) respectively. A hydrogen bond network between Sir2 and the S-alkylamidate (colored in pink) intermediate stabilize the observed conformation of the S-alkylamidate intermediate (C). Distances measured in angstroms between atoms of the S-alkylamidate and Sir2Tm H116 are displayed in panel (D).
Figure 4
Figure 4. Comparison of the Sir2Tm-Michaelis and Sir2Tm-S-alkylamidate complexes
The Sir2Tm-Michaelis structure (colored blue) was superimposed on to the Sir2Tm-S-alkylamidate structure (colored in gray). Only small movements of the NAD+ N-ribose and acetyl lysine in the Sir2 active site are necessary to form the S-alkylamidate, or the naturally occurring O-alkylamidate intermediate.
Figure 5
Figure 5. A conserved cluster of phenylalanines protect the peptidyl –imidate intermediate from hydrolysis and base exchange with nicotinamide
A conserved patch of bulky aromatic residues, colored in red, including Phe33, 48, and 162 shield the S-alkylamidate intermediate from solvent. (B) Sir2Tm Phe33 reorients from its Michaelis complex position (colored in blue) to stack against the S-alkylamidate intermediate (colored in gray). (C) The conformation of Phe 33 in the Sir2Tm-S-alkylamidate intermediate (colored in pink) is similar to its position in the Sir2 products structure (colored in green). In this conformation, Phe33 would protect the S-alkylamidate from base-exchange with nicotinamide.
Figure 6
Figure 6. Enzymatic characterization of the Sir2Tm F33A enzyme
Sir2Tm F33A (red curve) NAD+ consumption was measured and compared to wild type Sir2Tm enzyme (blue curve) (A). The Kcat of the Mutant enzyme, 0.3 sec−1, was 30 fold lower than wild type enzyme, 5.9 sec−1. To determine if Sir2Tm F33A enzyme was more sensitive to nicotinamide inhibition, base exchange assays with [14C] nicotinamide were preformed (B). Reaction products were separated on a silica TLC plate. As observed, the Sir2Tm F33A protein has higher base exchange activity. (C) Nicotinamide inhibition assays were preformed, and Sir2Tm F33A,IC50 of 0.1 μM, is at least three orders of magnitude more sensitive to nicotinamide inhibition than the wild type enzyme, IC50 of 480 μM.
Figure 6
Figure 6. Enzymatic characterization of the Sir2Tm F33A enzyme
Sir2Tm F33A (red curve) NAD+ consumption was measured and compared to wild type Sir2Tm enzyme (blue curve) (A). The Kcat of the Mutant enzyme, 0.3 sec−1, was 30 fold lower than wild type enzyme, 5.9 sec−1. To determine if Sir2Tm F33A enzyme was more sensitive to nicotinamide inhibition, base exchange assays with [14C] nicotinamide were preformed (B). Reaction products were separated on a silica TLC plate. As observed, the Sir2Tm F33A protein has higher base exchange activity. (C) Nicotinamide inhibition assays were preformed, and Sir2Tm F33A,IC50 of 0.1 μM, is at least three orders of magnitude more sensitive to nicotinamide inhibition than the wild type enzyme, IC50 of 480 μM.
Figure 7
Figure 7. HPLC analysis of Sir2Tm reactions
The Sir2Tm (green) and Sir2Tm F33A reactions (red) were analyzed by reverse phase HPLC. Standards were run to identify reaction components (Blue trace)
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References

    1. Armstrong CM, Kaeberlein M, Imai SI, Guarente L. Mutations in Saccharomyces cerevisiae gene SIR2 can have differential effects on in vivo silencing phenotypes and in vitro histone deacetylation activity. Mol Biol Cell. 2002;13:1427–1438. - PMC - PubMed
    1. Avalos JL, Bever KM, Wolberger C. Mechanism of sirtuin inhibition by nicotinamide: altering the NAD(+) cosubstrate specificity of a Sir2 enzyme. Mol Cell. 2005;17:855–868. - PubMed
    1. Avalos JL, Boeke JD, Wolberger C. Structural basis for the mechanism and regulation of Sir2 enzymes. Mol Cell. 2004;13:639–648. - PubMed
    1. Avalos JL, Celic I, Muhammad S, Cosgrove MS, Boeke JD, Wolberger C. Structure of a Sir2 enzyme bound to an acetylated p53 peptide. Mol Cell. 2002;10:523–535. - PubMed
    1. Bitterman KJ, Anderson RM, Cohen HY, Latorre-Esteves M, Sinclair DA. Inhibition of silencing and accelerated aging by nicotinamide, a putative negative regulator of yeast sir2 and human SIRT1. J Biol Chem. 2002;277:45099–45107. - PubMed

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