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. 2000 Dec 19;97(26):14178-82.
doi: 10.1073/pnas.250422697.

Silent information regulator 2 family of NAD- dependent histone/protein deacetylases generates a unique product, 1-O-acetyl-ADP-ribose

K G Tanner  1 , J Landry, R Sternglanz, J M Denu
Affiliations

Silent information regulator 2 family of NAD- dependent histone/protein deacetylases generates a unique product, 1-O-acetyl-ADP-ribose

K G Tanner et al. Proc Natl Acad Sci U S A. .

Abstract

Conflicting reports have suggested that the silent information regulator 2 (SIR2) protein family employs NAD(+) to ADP-ribosylate histones [Tanny, J. C., Dowd, G. J., Huang, J., Hilz, H. & Moazed, D. (1999) Cell 99, 735-745; Frye, R. A. (1999) Biochem. Biophys. Res. Commun. 260, 273-279], deacetylate histones [Landry, J., Sutton, A., Tafrov, S. T., Heller, R. C., Stebbins, J., Pillus, L. & Sternglanz, R. (2000) Proc. Natl. Acad. Sci. USA 97, 5807-5811; 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. (2000) Proc. Natl. Acad. Sci. USA 97, 6658-6663], or both [Imai, S., Armstrong, C. M., Kaeberlein, M. & Guarente, L. (2000) Nature (London) 403, 795-800]. Uncovering the true enzymatic function of SIR2 is critical to the basic understanding of its cellular function. Therefore, we set out to authenticate the reaction products and to determine the intrinsic catalytic mechanism. We provide direct evidence that the efficient histone/protein deacetylase reaction is tightly coupled to the formation of a previously unidentified acetyl-ADP-ribose product (1-O-acetyl-ADP ribose). One molecule of NAD(+) and one molecule of acetyl-lysine are readily catalyzed to one molecule of deacetylated lysine, nicotinamide, and 1-O-acetyl-ADP-ribose. A unique reaction mechanism involving the attack of enzyme-bound acetate or the direct attack of acetyl-lysine on an oxocarbenium ADP-ribose intermediate is proposed. We suggest that the reported histone/protein ADP-ribosyltransferase activity is a low-efficiency side reaction that can be explained through the partial uncoupling of the intrinsic deacetylation and acetate transfer to ADP-ribose.

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Figures

Figure 1
Figure 1
HPLC analysis of the HST2 NAD-dependent deacetylation reaction. (A) Reverse-phase HPLC elution of substrate standards (NAD+ and AcLys-14 H3 peptide) and of potential products (H3 peptide, acetate, ADP-ribose, and nicotinamide). Approximately 1.5 nmol AcLys-14 H3 peptide, 1.0 nmol H3 peptide, 10 nmol NAD, 15 nmol ADP-ribose, and 15 nmol nicotinamide were mixed and subjected to reverse-phase chromatography. In a separate HPLC run, 1.0 nmol sodium [3H]acetate (100,000 cpm/nmol) was subjected to chromatography. Order of elution: nicotinamide, acetate, ADP-ribose, NAD+, H3 peptide, and AcLys-14 H3 peptide. The elution position of acetate (∗) was determined by using authentic [3H]acetate and detection by liquid scintillation counting. All others were detected by UV absorption at 214 nm. (B) Elution of products from the HST2 NAD-dependent deacetylation reaction, detected by UV absorbance at 214 nm. ∗, Previously unidentified product. Conditions: 3.6 μM HST2/175 μM NAD+/525 μM Lys-14 AcH3/1 mM DTT for 30 min at 37°C before quenching with TFA to final concentration of 1%. (C) Amount of deacetylation correlates exactly with the consumption of NAD+. This graph displays the progress curves of deacetylation at fixed but limiting [NAD+]. HST2 reaction was allowed to proceed to completion under various limiting [NAD+], and the amount of deacetylated H3 peptide was determined. Conditions: 375 nM HST2/175 μM Lys-14 AcH3/8.75, 17.5, or 35 μM NAD+/1 mM DTT for 20 min at 37°C before quenching with TFA to 1%. (D) Steady-state rate of nicotinamide and deacetylated H3 peptide formation at various fixed [NAD+]. Conditions: 375 nM HST2/175 μM Lys-14 AcH3/8.75 μM–280 μM NAD+/1 mM DTT for 1 min at 37°C before quenching with TFA to a final concentration of 1%.
Figure 2
Figure 2
Acetate is not a primary product of HST2-catalyzed histone/protein deacetylation. H3 peptide was stoichiometrically acetylated on Lys-14 by the histone acetyltransferase P/CAF and [3H]AcCoA. Monoacetylated [3H]AcLys-14 H3 peptide was then purified by HPLC (A) and used as a substrate in the HST2 deacetylation reactions (B). On complete consumption of [3H]AcLys-14 H3 by HST2, all of the original 3H from H3 peptide was converted to a labeled product that eluted much later than authentic acetate. Conditions: 375 nM HST2/175 μM NAD+/5 μM [3H]Lys-14 AcH3/1 mM DTT for 1 min at 37°C before quenching with TFA to a final concentration of 1%. (C) Single turnover rapid quench-flow analysis. HST2 (10 μM) and NAD+ (300 μM) were mixed rapidly with 2.5 μM [3H]AcLys-14 H3 peptide at 22 ± 3°C (pH 7.5) in a Hi-Tech rapid quench-flow device, RQF-63. Between 31 ms and 8 s, reactions were quenched with TFA (1%). Quantification of [3H]AcLys-14 H3 peptide (⋄) and the [3H]acetate adduct (○) was accomplished by liquid scintillation counting of these species separated by using reverse-phase HPLC. Data were fitted to a single exponential, with yielding rate constants of 2.0 ± 0.1 s−1 for [3H]AcLys-14 H3 peptide deacetylation and 2.3 ± 0.2 s−1 for [3H]acetate adduct formation.
Figure 3
Figure 3
Mass spectral analysis of the previously unknown product generated by HST2-catalyzed histone/protein deacetylation. MALDI mass spectrometry was used to identity a mass of 602 m/z, consistent with the formation of acetyl-ADP- ribose (1-O-acetyl-ADP-ribose). For comparison, authentic ADP-ribose yielded a predicted mass of 560 m/z. With both ADP-ribose and acetyl-ADP-ribose, masses corresponding to the association of one and two sodium ions also were observed.
Figure 4
Figure 4
Proposed catalytic mechanisms for the production of 1-O-acetyl-ADP-ribose. (A) Attack of transiently formed acetate on an oxocarbenium ADP-ribose intermediate. Nicotinamide elimination from NAD+ to produce an oxocarbenium ADP-ribose intermediate is coupled to acetyl-lysine binding or hydrolysis. The enzyme-bound acetate generated in the deacetylation reaction attacks the oxocarbenium cation to produce 1-O-acetyl-ADP-ribose. (B) Acetyl-lysine condenses directly with the oxocarbenium cation. After the formation of the oxocarbenium cation as in A, the acyl oxygen of acetyl-lysine condenses with the oxocarbenium cation. A hydroxide ion then attacks this intermediate to form a tetravalent intermediate, which can collapse to produce 1-O-acetyl-ADP-ribose through the use of enzyme general acid/base catalysis. With either mechanism (A or B), the chemistry could occur in either stepwise or concerted fashion. For clarity, we have drawn the chemical events as stepwise events.
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