An NAD⁺‑Dependent Sirtuin Depropionylase and Deacetylase (Sir2La) from the Probiotic Bacterium <i>Lactobacillus acidophilus</i> NCFM

Sirtuins, a group of NAD⁺-dependent deacylases, have emerged as the key connection between NAD⁺ metabolism and aging. This class of enzymes hydrolyzes a range of ε-<i>N</i>-acyllysine PTMs, and determining the repertoire of catalyzed deacylation reactions is of high importance to fully elucidate the roles of a given sirtuin. Here we have identified and produced two potential sirtuins from the probiotic bacterium <i>Lactobacillus acidophilus</i> NCFM. Screening more than 80 different substrates, covering 26 acyl groups on five peptide scaffolds, demonstrated that one of the investigated proteins, Sir2La, is a <i>bona fide</i> NAD⁺-dependent sirtuin, catalyzing hydrolysis of acetyl-, propionyl-, and butyryllysine. Further substantiating the identity of Sir2La as a sirtuin, known sirtuin inhibitors, nicotinamide and suramin, as well as a thioacetyllysine compound inhibit the deacylase activity in a concentration-dependent manner. On the basis of steady-state kinetics, Sir2La showed a slight preference for propionyllysine (Kpro) over acetyllysine (Kac). For nonfluorogenic peptide substrates, the preference is driven by a remarkably low <i>K</i>_M (280 nM vs 700 nM, for Kpro and Kac, respectively), whereas <i>k</i>_cat was similar (21 × 10^–3 s^–1). Moreover, while NAD⁺ is a prerequisite for Sir2La-mediated deacylation, Sir2La has a very high <i>K</i>_M for NAD⁺ compared to the expected levels of the dinucleotide in <i>L. acidophilus</i>. Sir2La is the first sirtuin from Lactobacillales and of the Gram-positive bacterial subclass of sirtuins to be functionally characterized. The ability to hydrolyze propionyl- and butyryllysine emphasizes the relevance of further exploring the role of other short-chain acyl moieties as PTMs