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. 2008 Sep 23;47(38):10227-39.
doi: 10.1021/bi800767t. Epub 2008 Aug 26.

Plasmodium falciparum Sir2 is an NAD+-dependent deacetylase and an acetyllysine-dependent and acetyllysine-independent NAD+ glycohydrolase

Jarrod B French  1 , Yana Cen, Anthony A Sauve
Affiliations

Plasmodium falciparum Sir2 is an NAD+-dependent deacetylase and an acetyllysine-dependent and acetyllysine-independent NAD+ glycohydrolase

Jarrod B French et al. Biochemistry. .

Abstract

Sirtuins are NAD (+)-dependent enzymes that deacetylate a variety of cellular proteins and in some cases catalyze protein ADP-ribosyl transfer. The catalytic mechanism of deacetylation is proposed to involve an ADPR-peptidylimidate, whereas the mechanism of ADP-ribosyl transfer to proteins is undetermined. Herein we characterize a Plasmodium falciparum sirtuin that catalyzes deacetylation of histone peptide sequences. Interestingly, the enzyme can also hydrolyze NAD (+). Two mechanisms of hydrolysis were identified and characterized. One is independent of acetyllysine substrate and produces alpha-stereochemistry as established by reaction of methanol which forms alpha-1- O-methyl-ADPR. This reaction is insensitive to nicotinamide inhibition. The second solvolytic mechanism is dependent on acetylated peptide and is proposed to involve the imidate to generate beta-stereochemistry. Stereochemistry was established by isolation of beta-1- O-methyl-ADPR when methanol was added as a cosolvent. This solvolytic reaction was inhibited by nicotinamide, suggesting that nicotinamide and solvent compete for the imidate. These findings establish new reactions of wildtype sirtuins and suggest possible mechanisms for ADP-ribosylation to proteins. These findings also illustrate the potential utility of nicotinamide as a probe for mechanisms of sirtuin-catalyzed ADP-ribosyl transfer.

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Figures

Figure 1
Figure 1. Deacetylation of peptide substrates catalyzed by Pf-Sir2
(A) HPLC chromatograms showing the deacetylation of acetylated-H3 by Pf-Sir2 and SirT1 over time. (B) Deacetylation of H4. (C) Deacetylation of p300. (D)Saturation curves for steady-state deacetylation rates for the three different peptides.
Figure 2
Figure 2. Nicotinamide Base-Exchange Catalyzed by Pf-Sir2
(A) and (B): Inhibition of the deacetylation reaction by increasing concentrations of nicotinamide. (A) 500 ∝M H3 peptide, 250 ∝M NAD+ and 150 mM phosphate buffer, pH 7.3, quantified by integration of deacetylated product peaks in the HPLC chromatograms. (B) 200 ∝M p300 peptide, 400 ∝M NAD+ and 150 mM phosphate buffer, pH 7.5, and quantified by integration of deacetylated product peaks in the HPLC chromatograms. (C) and (D): Kinetics of the base-exchange chemistry catalyzed by Pf-Sir2 as measured by exchange of [carbonyl-14C]-nicotinamide into unlabeled NAD+. (C) 200 ∝M H3 peptide, 400 ∝M NAD+ and 150 mM phosphate buffer, pH 7.5. (D) 200 ∝M p300 peptide, 400 ∝M NAD+ and 150 mM phosphate buffer, pH 7.5.
Figure 3
Figure 3. Thionicotinamide Base-Exchange Catalyzed by Pf-Sir2
(A) HPLC chromatograms showing the thionicotinamide base-exchange reaction and negative control. (B) Steady-state saturation kinetics of the thionicotinamide base-exchange reaction as catalyzed by Pf-Sir2 in the presence of 400 ∝M NAD+ and 400 ∝M H3 peptide and increasing concentrations of thionicotinamide.
Figure 4
Figure 4. Hydrolysis of NAD+ Catalyzed by Pf-Sir2
(A) HPLC chromatograms showing the production of ADPR by SirT1 and Pf-Sir2 under similar conditions. (B) the kinetics of the hydrolysis reaction for varying concentrations of peptide carried out in the presence of 400 ∝M NAD+ and 150 mM phosphate buffer, pH 7.3.
Figure 5
Figure 5. Production of α-face Methanolysis Product
(A) HPLC chromatograms showing the α-MeOADPR standard, a Pf-Sir2 reaction run without peptide and in the presence of 30% MeOH, and a control reaction containing no enzyme run in the presence of 30% MeOH. (B) MALDI-MS data showing the m/z values for the methanolysis products collected from the Pf-Sir2 catalyzed reactions in the presence of 30% CH3OH and 30% CD3OH respectively.
Figure 6
Figure 6. Production of β-face Methanolysis Product
(A) HPLC chromatograms showing the β-MeOADPR standard, and a Pf-Sir2 reaction run in the presence of H3 peptide and 30% MeOH. (B) Bottom: Pf-Sir2 catalyzed methanolysis with different concentrations of H3 Productions of α-MeOADPR (circle) and β-MeOADPR (square) with different concentrations of H3 are shown. The reactions contained 400 ∝M of NAD+ in 100 mM phosphate buffer with 0, 10, 25, 50, 75, 100, 150, 250, 400 and 500 ∝M of H3 all at pH 8.5. The following Michaelis parameters were determined by fitting the curves with KaleidaGraph: Ki (α-MeO-ADPR) = 70 ∝M, k0 = 2.7 × 10−4 s−1 Km (β-MeO-ADPR) = 119 ∝M, k500 = 1.6 × 10−4 s−1 ksolvolysis ([H3] = 0) = 1.38 × 10−3 s−1, ksolvolysis ([H3] = 500 ∝M) = 1.40 × 10−3 s−1
Figure 7
Figure 7. Hydrolysis is Incompletely Inhibited by Nicotinamide
(A) The effect of nicotinamide on the hydrolytic activity of Pf-Sir2 in the presence and absence of peptide. The curve fit to the data for hydrolysis in increasing P300 concentrations (solid circles) is shown alongside the hydrolysis data obtained by running the reactions in 400 ∝M P300 and increasing nicotinamide concentrations (circles in squares). The hydrolysis rate is incompletely inhibited and plateaus at a level equal to the rate of peptide independent hydrolysis (dashed line). The data for peptide independent hydrolysis in varying nicotinamide concentrations is shown (triangles) and is fit by a straight line (dashed line). (B) The plot of the ratio of β to α stereochemistry of methanolysis products with increasing nicotinamide concentration as determined by HPLC. The plotted points were fit to a curve of form r = r0 – rmax ([I]/(Ki +[I])) where r is the ratio of stereochemistry observed for a given concentration of nicotinamide, r0 is the observed stereochemical ratio when no nicotinamide is present, rmax is the maximal suppression of the ratio, Ki is the apparent inhibition constant of nicotinamide and [I] is the concentration of nicotinamide. The HPLC chromatograms (inset) illustrate that when nicotinamide is added there is a decrease in the β–1-O-methylADPR peak, but there is little change in the production of α–1-O-methylADPR. The nicotinamide visible in the lower trace was produced by the normal hydrolysis and methanolysis of NAD+ catalyzed by Pf-Sir2.
Scheme 1
Scheme 1
Reactions of acetylysine peptides in base exchange and deacetylation pathways. Rate constants for deacetylation and base exchange are shown for the respective steps.
Scheme 2
Scheme 2
Proposed reaction of NAD+ in active site of Pf-Sir2 with either solvent of acetyllysine.
Scheme 3
Scheme 3
Reaction choices of solvolysis with inversion of stereochemistry or competition reaction of acetyllysine to form imidate which can react with solvent to form product with overall retention of stereochemistry.
Scheme 4
Scheme 4
Overall reaction scheme of Pf-Sir2 catalyzed reactions. Top scheme depicts solvolytic chemistry in the absence of peptide which gives inversion of stereochemistry (reaction a). Bottom scheme depicts acetyllysine dependent chemistries which occur via the imidate including base exchange (reaction b), solvolysis from the imidate (reaction c) and deacetylation (reaction d)
Scheme 5
Scheme 5
Proposed general mechanisms of ADP-ribosyltransfer catalyzed by sirtuins as determined by chemistries reported in this study. Top chemistry is acetyllysine independent, gives inversion of stereochemistry versus NAD+ and is insensitive to nicotinamide inhibition. Bottom chemistry scheme shows ADP-ribosyltransfer from the imidate complex, to produce ADP-riboysltransfer with retention of stereochemistry versus NAD+. This reaction is predicted to be sensitive to nicotinamide inhibition.
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