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. 2011 Jun 17;286(24):21110-7.
doi: 10.1074/jbc.M111.237636. Epub 2011 Apr 17.

Hydrolysis of O-acetyl-ADP-ribose isomers by ADP-ribosylhydrolase 3

Atsushi Kasamatsu  1 , Motoyuki Nakao, Brian C Smith, Lindsay R Comstock, Tohru Ono, Jiro Kato, John M Denu, Joel Moss
Affiliations

Hydrolysis of O-acetyl-ADP-ribose isomers by ADP-ribosylhydrolase 3

Atsushi Kasamatsu et al. J Biol Chem. .

Abstract

O-acetyl-ADP-ribose (OAADPr), produced by the Sir2-catalyzed NAD(+)-dependent histone/protein deacetylase reaction, regulates diverse biological processes. Interconversion between two OAADPr isomers with acetyl attached to the C-2″ and C-3″ hydroxyl of ADP-ribose (ADPr) is rapid. We reported earlier that ADP-ribosylhydrolase 3 (ARH3), one of three ARH proteins sharing structural similarities, hydrolyzed OAADPr to ADPr and acetate, and poly(ADPr) to ADPr monomers. ARH1 also hydrolyzed OAADPr and poly(ADPr) as well as ADP-ribose-arginine, with arginine in α-anomeric linkage to C-1″ of ADP-ribose. Because both ARH3- and ARH1-catalyzed reactions involve nucleophilic attacks at the C-1″ position, it was perplexing that the ARH3 catalytic site would cleave OAADPr at either the 2″- or 3″-position, and we postulated the existence of a third isomer, 1″-OAADPr, in equilibrium with 2″- and 3″-isomers. A third isomer, consistent with 1″-OAADPr, was identified at pH 9.0. Further, ARH3 OAADPr hydrolase activity was greater at pH 9.0 than at neutral pH where 3″-OAADPr predominated. Consistent with our hypothesis, IC(50) values for ARH3 inhibition by 2″- and 3″-N-acetyl-ADPr analogs of OAADPr were significantly higher than that for ADPr. ARH1 also hydrolyzed OAADPr more rapidly at alkaline pH, but cleavage of ADP-ribose-arginine was faster at neutral pH than pH 9.0. ARH3-catalyzed hydrolysis of OAADPr in H(2)(18)O resulted in incorporation of one (18)O into ADP-ribose by mass spectrometric analysis, consistent with cleavage at the C-1″ position. Together, these data suggest that ARH family members, ARH1 and ARH3, catalyze hydrolysis of the 1″-O linkage in their structurally diverse substrates.

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Figures

FIGURE 1.
FIGURE 1.
Schematic diagram of substrates hydrolyzed by ARH1 and ARH3. A, structure of OAADPr with position of 1″-O-acetyl-linkage noted. B, structure of α-ADPr-arginine. C, Structure of poly(ADPr) (C1″-C2′).
FIGURE 2.
FIGURE 2.
pH-dependence of O-acetyl-ADP-ribose hydrolysis by ARH3. A–E, [14C]OAADPr (1 μm) (total volume, 200 μl) was incubated at pH 5.0 (A), 7.0 (B), or 9.0 (C) with (open circles) or without (filled circles) recombinant human ARH3 (1.5 pmol) for 1 h at 30 °C before separation of substrate and products as described under “Experimental Procedures.” Nonenzymatic hydrolysis of [14C]OAADPr was assayed in the same conditions at pH 7.0 (D) and 9.0 (E). Substrates and products were separated by RP-HPLC and radioactivity quantified by liquid scintillation counting. F, 1 μm [14C]OAADPr was incubated with ARH3 (1.5 pmol), 50 mm potassium phosphate (pH 5.0), 10 mm MgCl2, 5 mm DTT (total volume, 200 μl) for 35 min at pH 7.0 or 30 min at pH 5.0 (30 °C) followed by 5 min at pH 7.0 (pH was adjusted by the addition of 100 mm dipotassium hydrogen orthophosphate). Substrates and products were quantified as described above. The experiment was repeated three times with similar results.
FIGURE 3.
FIGURE 3.
Interconversion of O-acetyl-ADP-ribose isomers. A, OAADPr (peak A) was separated using RP-HPLC (elution time consistent with 3″-OAADPr) in 200 μl of water containing 0.05% (v/v) TFA as described under “Experimental Procedures.” B, purified 3″-OAADPr in reaction mix described in Fig. 1 at pH 9.0 was maintained at pH 9.0 and separated using RP-HPLC as described. C, purified 3″-OAADPr (pH 9.0) was incubated for 5 min at 30 °C followed by the addition of 0.05% (v/v) TFA and incubation for an additional 10 min at 30 °C before RP-HPLC analysis. The experiment was repeated three times with similar results.
FIGURE 4.
FIGURE 4.
Mass spectrometry of O-acetyl-ADP-ribose isomers. Separated isomers (A, B, and C) of OAADPr (1 μm) like that shown in Fig. 1C were subjected to MALDI-TOF mass spectrometry to reveal a peak at 600 m/z, consistent with the predicted mass of OAADPr, in each sample. The experiment was repeated three times with similar results.
FIGURE 5.
FIGURE 5.
ADP-ribose, 2″-N-acetyl-ADP-ribose, and 3″-N-acetyl-ADP-ribose inhibition of ARH3 activity. Human ARH3 (83.5 nm; 16.7 pmol) and 12.5 μm [3H]OAADPr with ADPr (open circles), 2″-N-acetyl-ADPr (open squares), or 3″-N-acetyl-ADPr (filled squares) were incubated for 30 min at 30 °C as described in Fig. 1 before quantification of [3H]acetate as described under “Experimental Procedures.”
FIGURE 6.
FIGURE 6.
pH-dependence of O-acetyl-ADP-ribose hydrolysis by ARH3 and ARH1. Human ARH 3 (1.5 pmol) or ARH1 (375 pmol) was incubated for 5 min with 1 μm OAADPr at the indicated pH as in Fig. 4 before RP-HPLC separation and quantification of ADPr. Blank data without enzyme were subtracted from each of these points. The experiment was repeated three times with similar results.
FIGURE 7.
FIGURE 7.
pH-dependence of α-ADP-ribosyl-arginine hydrolysis by ARH1. A, 25 μm α-ADP-ribosyl-[14C]arginine was incubated in 50 mm potassium phosphate buffer (pH 7.0), 10 mm MgCl2, and 5 mm DTT for 1 h at 30 °C with (dashed lines) or without (solid lines) recombinant human ARH1 (3 pmol) before RP-HPLC separation of substrate and products. Substrate and products were detected by absorbance at 254 nm. B, α-ADP-ribosyl-[14C]arginine was incubated as in Fig. 6A, with ARH1 (3 pmol) for the indicated time at 30 °C before quantification of ADPr. Results were similar in three experiments.
FIGURE 8.
FIGURE 8.
ARH3-catalyzed 18O labeling of ADPr. A, OAADPr (5 nmol) was incubated with ARH3 (1 nmol) in 40 mm pyridine buffer adjusted to pH 7.0 with formic acid, 1 mm MgCl2, and 90% H218O for 30 min at 30 °C. Then the mixture was diluted with methanol and subjected to mass spectrometry as described under “Experimental Procedures.” B and C, m/z = 558.15 corresponds to ADPr, and m/z = 560.15 to ADPr containing 18O atom. 18O incorporation was measured by the relative peak heights at each m/z. As a control, ADPr was incubated with heat-denatured ARH3 in the same mixture for 0 min (B) to confirm the natural distribution of heavy isotope or 30 min (C) to assess nonenzymatic exchange of the 1″-hydroxyl at 30 °C. Detection and measurement of 18O incorporation are described under “Experimental Procedures.” The experiment was repeated three times with similar results. The proposed reaction mechanisms for 18O incorporation are indicated in the upper panel.
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