Human SIRT1 multi-specificity is modulated by active-site vicinity substitutions during natural evolution.

Many enzymes that catalyze protein post-translational modifications (PTM) can specifically modify multiple target proteins. However, little is known regarding the molecular basis and evolution of multi-specificity in these enzymes. Here, we used a combined bioinformatics and experimental approaches to investigate the evolution of multi-specificity in the sirtuin-1 (SIRT1) deacetylase. Guided by bioinformatics analysis of SIRT1 orthologues and substrates, we identified and examined important amino acid substitutions that have occurred during the evolution of sirtuins in Metazoa and Fungi. We found that mutation of human SIRT1 at these positions, based on sirtuin orthologues from Fungi, could alter its substrate specificity. These substitutions lead to reduced activity toward K382 acetylated p53 protein, which is only present in Metazoa, without affecting the high activity toward the conserved histone substrates. Results from ancestral sequence reconstruction are consistent with a model in which ancestral sirtuin proteins exhibited multi-specificity, suggesting that the multi-specificity of some metazoan sirtuins, such as hSIRT1, could be a relatively ancient trait.