Substrate Specificity and Kinetic Mechanism of the NAD+-Dependent Deacylase from Mycobacterium tuberculosis, Rv1151c (Mt-Sirt).

The pathogen Mycobacterium tuberculosis (Mtb) relies on extensive metabolic remodeling and post-translational regulation of its proteins to retain persistence in its host, yet these biochemical functions remain largely uncharacterized, including the actions of lysine deacylases. Here, we characterized Rv1151c, the sole annotated NAD+-dependent deacylase in Mtb, which we designate Mt-Sirt. Using synthetic substrates, peptide libraries, and mutational analysis, we showed that Mt-Sirt exhibits strong selectivity for peptide substrates containing Nε-succinyl-lysine and long-chain fatty-acyl-lysine groups. Substitution of conserved active-site residues (Y53F and R56M) confirmed their roles in substrate recognition and acyl-group discrimination. Inhibition studies revealed that nicotinamide, NADH, ADP-ribose, and mechanism-based thioamide/thiourea analogs inhibited Mt-Sirt activity with low-micromolar to nanomolar potency. Initial-velocity, product-inhibition, and dead-end inhibition analyses confirmed that Mt-Sirt operates by an equilibrium-ordered kinetic mechanism in which NAD+ binds first, followed by the Nε-acylated peptide substrate. We further demonstrated that chemically succinylated Mtb isocitrate lyase 1 (ICL1), a key enzyme in Mtb metabolism, was a substrate of Mt-Sirt in vitro. Lastly, metabolic labeling with tetradec-13-ynoic acid (Alk-12) revealed widespread lysine and N-terminal fatty acylation in Mtb cells, suggesting that Mt-Sirt may regulate previously unrecognized fatty-acyl PTMs in vivo. Together, these findings provide a kinetic and substrate-specificity analysis of Mt-Sirt, and highlight its potential role in controlling reversible lysine acylation in Mtb.