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J. Biol. Chem., Vol. 283, Issue 9, 5317-5326, February 29, 2008

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Acetylation-dependent ADP-ribosylation by Trypanosoma brucei Sir2^*

From the Department of Biomolecular Chemistry, University of Wisconsin, School of Medicine and Public Health, Madison, Wisconsin 53706

Sirtuins are a highly conserved family of proteins implicated in diverse cellular processes such as gene silencing, aging, and metabolic regulation. Although many sirtuins catalyze a well characterized protein/histone deacetylation reaction, there are a number of reports that suggest protein ADP-ribosyltransferase activity. Here we explored the mechanisms of ADP-ribosylation using the Trypanosoma brucei Sir2 homologue TbSIR2rp1 as a model for sirtuins that reportedly display both activities. Steady-state kinetic analysis revealed a highly active histone deacetylase (k_cat = 0.1 s^–1, with K_m values of 42 µM and for NAD⁺ and 65 µM for acetylated substrate). A series of biochemical assays revealed that TbSIR2rp1 ADP-ribosylation of protein/histone requires an acetylated substrate. The data are consistent with two distinct ADP-ribosylation pathways that involve an acetylated substrate, NAD⁺ and TbSIR2rp1 as follows: 1) a noncatalytic reaction between the deacetylation product O-acetyl-ADP-ribose (or its hydrolysis product ADP-ribose) and histones, and 2) a more efficient mechanism involving interception of an ADP-ribose-acetylpeptide-enzyme intermediate by a side-chain nucleophile from bound histone. However, the sum of both ADP-ribosylation reactions was 5 orders of magnitude slower than histone deacetylation under identical conditions. The biological implications of these results are discussed.

Received for publication, September 11, 2007 , and in revised form, December 3, 2007.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S3.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed: Dept. of Biomolecular Chemistry, University of Wisconsin, School of Medicine and Public Health, 1300 University Ave, Madison, WI 53706. Tel.: 608-265-1859; Fax: 608-262-5253; E-mail: jmdenu{at}wisc.edu.

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