Acetylation-dependent ADP-ribosylation by Trypanosoma brucei Sir2.
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.
Kowieski
Terri M
TM
Department of Biomolecular Chemistry, University of Wisconsin, School of Medicine and Public Health, Madison, Wisconsin 53706, USA.
Lee
Susan
S
Denu
John M
JM
D000107:Acetylation; D000818:Animals; D002384:Catalysis; D006655:Histone Deacetylases / Q000737:chemistry* / Q000378:metabolism; D006657:Histones / Q000737:chemistry* / Q000378:metabolism; D009243:NAD / Q000737:chemistry* / Q000378:metabolism; D015800:Protozoan Proteins / Q000737:chemistry* / Q000378:metabolism; D037761:Sirtuins / Q000737:chemistry* / Q000378:metabolism; D014346:Trypanosoma brucei brucei / Q000201:enzymology*; D014982:Xenopus laevis
D006657:Histones; D015800:Protozoan Proteins; D009243:NAD; D037761:Sirtuins; D006655:Histone Deacetylases