Structural studies of Sir2 enzyme mechanism

Sir2 enzymes, also known as sirtuins, comprise an ancient family of NAD +-dependent protein deacetylases. Sirtuins are involved in a broad range of biological processes, including transcriptional silencing, DNA repair, DNA recombination, DNA replication, genomic stability, apoptosis, cell cycle, metabolism and the process of aging. They specifically deacetylate acetyl-lysine residues of target proteins and in the process cleave NAD+ to produce acetylated ADP-ribose and nicotinamide. The latter acts as a non-competitive inhibitor of the reaction and is very important in the regulation of sirtuins in cellular processes. This thesis describes biochemical and biophysical studies that address the structure and function of this intriguing family of enzymes. It contains several crystal structures of sirtuins in different ligated states, including acetylated peptide, NAD+ in productive and non-productive conformations, ADP-ribose, acetyl-lysine mimics (polyethylene glycol and polypropylene glycol) and the apo-enzyme. Comparison between these and other structures reveals that sirtuins undergo significant conformational changes during the catalytic cycle. The acetylated peptide binds in a cleft between two domains of the enzyme, making an enzyme-substrate beta-sheet. In the absence of acetyl-lysine, NAD+ binds in several low-energy, non-productive conformations; but binding of acetyl-lysine seems to trigger a shift in the NAD+ to a high-energy conformation that destabilizes its ground state, making it productive for catalysis. The mechanism of sirtuin regulation by nicotinamide and its effect as a non-competitive inhibitor of the deacetylation reaction is also addressed in this work.