Summary
Sirtuins are NAD-dependent deacylases. Previous studies have established two important enzymatic intermediates in sirtuin-catalyzed deacylation, an alkylamidate intermediate I, which is then converted to a bicyclic intermediate II. However, how intermediate II is converted to products is unknown. Based on potent SIRT2-specific inhibitors we developed, here we report crystal structures of SIRT2 in complexes with a thiomyristoyl (TM) inhibitor and carba-NAD or NAD. Interestingly by soaking crystals with NAD, we capture a distinct covalent catalytic intermediate (III) that is different from the intermediates I and II. In this intermediate, the covalent bond between the S and the myristoyl carbonyl carbon is broken and we believe this intermediate III is the decomposition product of II in route to form the end products. MALDI-TOF data further support the intermediate III formation. This is the first time such an intermediate is captured by X-ray crystallography and provides more mechanistic insights into sirtuin-catalyzed reactions.
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