Deacylation Mechanism by SIRT2 Revealed in the 1′-SH-2′-O-Myristoyl Intermediate Structure

Sirtuins are NAD-dependent deacylases, and previous studies have identified two key enzymatic intermediates in the sirtuin-catalyzed deacylation process: an alkylamidate intermediate (I), which is then converted into a bicyclic intermediate (II). However, the mechanism by which intermediate II is converted into the final products remains unclear. In this study, we present crystal structures of SIRT2 in complex with a thiomyristoyl lysine peptide-based inhibitor and either carba-NAD or NAD, based on potent SIRT2-specific inhibitors we developed. Remarkably, by soaking the crystals with NAD, we were able to capture a novel covalent catalytic intermediate (III), distinct from the previously identified intermediates I and II. In this new intermediate, the covalent bond between the sulfur atom and the myristoyl carbonyl carbon is broken, suggesting that intermediate III may be a decomposition product of II, leading toward the formation of the end products. MALDI-TOF data further corroborate the presence of intermediate III. This is the first time such an intermediate has been captured using X-ray crystallography, providing valuable mechanistic insights into the sirtuin-catalyzed reaction process.