Satoko Akashi

Sirtuin 2 (SIRT2) is a class III histone deacetylase that is highly conserved from bacteria to mammals. We prepared and characterized the wild‐type (WT) and mutant forms of the histone deacetylase (HDAC) domain of human SIRT2 ( h SIRT2) using various biophysical methods and evaluated their deacetylation activity. We found that WT h SIRT2 HDAC (residues 52–357) forms a homodimer in a concentration‐dependent manner with a dimer–monomer dissociation constant of 8.3 ± 0.5 μM, which was determined by mass spectrometry. The dimer was disrupted into two monomers by binding to the HDAC inhibitors SirReal1 and SirReal2. We also confirmed dimer formation of h SIRT2 HDAC in living cells using a NanoLuc complementation reporter system. Examination of the relationship between dimer formation and deacetylation activity using several mutants of h SIRT2 HDAC revealed that some non‐dimerizing mutants exhibited deacetylation activity for the N‐terminal peptide of histone H3, similar to the wild type. The h SIRT2 HDAC mutant Δ 292–306, which lacks a SIRT2‐specific disordered loop region, was identified to exist as a monomer with slightly reduced deacetylation activity; the X‐ray structure of the mutant Δ 292–306 was almost identical to that of the WT h SIRT2 HDAC bound to an inhibitor. These results indicate that h SIRT2 HDAC forms a dimer, but this is independent of deacetylation activity. Herein, we discuss insights into the dimer formation of h SIRT2 based on our biophysical experimental results.