SIRT3 differentially regulates lysine benzoylation from SIRT2 in mammalian cells

Lysine benzoylation (Kbz), a new type of protein post-translational modification (PTM) we discovered, has garnered significant attention. While we initially identified SIRT2 as a debenzoylase in mammalian cells, recent findings suggest its exclusivity may be questioned. However, other debenzoylases in mammalian cells remain underexplored. Here, our study reveals SIRT3 as an additional debenzoylase. Through quantitative analysis, we identified 1,075 Kbz sites in mammalian cells, with 44 specifically mediated by SIRT3 and 66 influenced by SIRT2. Notably, SIRT3 and SIRT2 regulate distinct Kbz substrates, indicating involvement in different cellular processes. Functional investigations demonstrated SIRT3’s regulation of benzoylated protein peptidyl-prolyl cis-trans isomerase F (PPIF), where K73bz and K197bz markedly diminished interactions with the tumor suppressor p53. Additionally, K978bz on ATP-citrate lyase (ACLY) notably inhibited its enzymatic activity. This study not only identifies a debenzoylase and its Kbz substrates but also enhances our understanding of Kbz’s biological functions.