Abstract
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 Km values of 42 microm and for NAD+ and 65 microm 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 approximately 5 orders of magnitude slower than histone deacetylation under identical conditions. The biological implications of these results are discussed.
Highlights
There is general consensus that many sirtuins affect biological pathways by catalyzing the NADϩ-dependent deacetylation of target proteins, a number of reports have suggested that some sirtuins catalyze protein ADP-ribosylation, either exclusively or in conjunction with their inherent deacetylase activity
The second pathway is responsible for the majority of TbSIR2rp1dependent ADPr transfer and involves a mechanism in which a side-chain nucleophile from bound histone attacks an intermediate from the catalytic pathway that normally leads to deacetylated protein and OAADPr
TbSIR2 displays histone deacetylation efficiencies that are comparable with other sirtuins that exhibit robust deacetylation activity [34, 47]; TbSIR2 ADP-ribosylation of histones was ϳ5 orders of magnitude slower than deacetylation
Summary
There is general consensus that many sirtuins affect biological pathways by catalyzing the NADϩ-dependent deacetylation of target proteins, a number of reports have suggested that some sirtuins catalyze protein ADP-ribosylation, either exclusively or in conjunction with their inherent deacetylase activity. Because TbSIR2 is matic contrast, TbSIR2 had a much lower capacity to transfer the an active histone deacetylase, we determined whether a portion label from [32P]NADϩ to recombinant purified histones
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