Side chain specificity of ADP-ribosylation by a sirtuin

Kamau Fahie, Po Hu, Stephen Swatkoski, Robert J. Cotter, Yingkai Zhang, Cynthia Wolberger

Research output: Contribution to journalArticlepeer-review

Abstract

Endogenous mono-ADP-ribosylation in eukaryotes is involved in regulating protein synthesis, signal transduction, cytoskeletal integrity, and cell proliferation, although few cellular ADP-ribosyltransferases have been identified. The sirtuins constitute a highly conserved family of protein deacetylases, and several family members have also been reported to perform protein ADP-ribosylation. We characterized the ADP-ribosylation reaction of the nuclear sirtuin homolog Trypanosoma brucei SIR2-related protein 1 (TbSIR2RP1) on both acetylated and unacetylated substrates. We demonstrated that an acetylated substrate is not required for ADP-ribosylation to occur, indicating that the reaction performed by TbSIR2RP1 is a genuine enzymatic reaction and not a side reaction of deacetylation. Biochemical and MS data showed that arginine is the major ADP-ribose acceptor for unacetylated substrates, whereas arginine does not appear to be the major ADP-ribose acceptor in reactions with acetylated histone H1.1. We performed combined ab initio quantum mechanical\molecular mechanical molecular dynamics simulations, which indicated that sirtuin ADP-ribosylation at arginine is energetically feasible, and involves a concerted mechanism with a highly dissociative transition state. In comparison with the corresponding nicotinamide cleavage in the deacetylation reaction, the simulations suggest that sirtuin ADP-ribosylation would be several orders slower but less sensitive to nicotinamide inhibition, which is consistent with experimental results. These results suggest that TbSIR2RP1 can perform ADP-ribosylation using two distinct mechanisms, depending on whether or not the substrate is acetylated.

Original languageEnglish (US)
Pages (from-to)7159-7176
Number of pages18
JournalFEBS Journal
Volume276
Issue number23
DOIs
StatePublished - Dec 2009

Keywords

  • ADP-ribose acceptor
  • ADP-ribosyltransferase
  • Mono-ADP-ribosylation
  • Sirtuin
  • TbSIR2

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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