Understanding the multi-specificity of SIRT1

SIRT1 is a post translational modifications (PTM) enzyme that deacetylates lysine residues in central cellular proteins to control signaling pathways. The yeast ortholog of SIRT1, Sir2, deacetylates only histones, while SIRT1 has hundreds of substrates such as histones, P53 and NFκB. To understand the molecular basis and the evolution of multi-specificity in SIRT1, we took three approaches. First, guided by bioinformatics analysis of SIRT1 orthologues and substrate repertoire expansion, we constructed and thoroughly examined SIRT1 variants containing two and four mutations located at the vicinity of the enzyme active site that originate from distantly related SIRT1 orthologues including those from yeast. Second, we used phylogenetic analysis and ancestral sequence reconstruction to guide mutagenesis of key specificity determining positions to test the impact of these residues on specificity. Third, we computationally designed 50 SIRT1 mutants containing several point mutations surrounding the active site. The mutations were designed to have minimum effect on the folding and stability of SIRT1. Each mutant in all three approaches was expressed and its activity was tested using three different assays including Flour de lys assay (FDL) using acetyl-lysine substrate, ammonia assay using unmodified acetylated peptides derived from SIRT1 substrates and activity assay with acetylated proteins including p53, RelA and histones. Our approach allowed us to identify active site residues in hSIRT1 that are essential for its multi-specificity and show evidence that multispecificity was already present in ancestral SIRT1 protein but was lost in some branches through evolution.