SIRT5 Mutants Reveal Insights into Sirtuin Regulation

Sirtuins are emerging as key regulators for metabolism and aging. These regulations were primarily attributed to sirtuin's NAD + dependent deacetylase activity. Among the seven human sirtuins, SIRT5 is the only one that also catalyzes desuccinylation. It is becoming clear that sirtuins have evolved to adapt different regulatory mechanisms of their activities. However, detailed studies on substrate specificity, isozyme selectivity, and inhibition mechanisms of sirtuins are still lacking. Two amino acids—Y102 and R105—are responsible for SIRT5′s desuccinylation activity. Therefore, we made single and double muatations on the two residues and compared their steady‐state kinetic features with those of wild type SIRT5 and SIRT1. We noticed that Y102 and R105 account partially for the differential substrate specificity and drug isoform selectivity of SIRT5, implying that sirtuin activity is also regulated by the dynamic flexibility in the protein structures. Our inhibition analysis showed that nicotinamide inhibited SIRT5′s desuccinylation activity by competing with NAD + ; while it inhibited SIRT1′s and SIRT5′s deacetylation activity via a combination of two pathways—competing with NAD + and base‐exchange. Combining it with our bisubstrate kinetic analysis, we conclude that the binding of succinylated substrate creates a tighter binding site for both NAD + and nicotinamide, generating alternative nicotinamide regulations of SIRT5. Our research studies provide insight into Sirtuin regulation and the rationale designing of sirtuin isoform selective inhibitors.