ABSTRACT
SIRT1, human ortholog of yeast SIR2 protein, deacetylates histones and several other transcription factors. Recently, SIRT1 has emerged as a drug target for treating age related diseases, type II diabetes, neurodegeneration, inflammation and cancer. Here, we have optimized production of functionally active wild type full-length SIRT1 protein and its N-terminal deleted mutants. In a comparative study, we found that the region containing 192-208 amino acids towards the N-terminus is critical for right conformational folding of the protein to retain its deacetylase activity. The EC(50) and IC(50) values obtained with standard modulators showed that the SRT(748) & SRT(556) can deacetylate substrate and are activated by resveratrol, whereas, deacetylase activity of all the other deletion mutants (SRT(540), SRT(532), SRT(507) and SRT(503)) was lost. We further report that the peptide substrate K(m) for SRT(748) (70+/-5.2 microM) was comparable to SRT(556) (93+/-5.4 microM). The K(m) for NAD(+) substrate was 176 & 274 microM for SRT(748) and SRT(556), respectively. Similar substrate affinity studies demonstrate that either of the protein (SRT(748) or SRT(556)) can be utilized for screening SIRT1 modulators. We have also examined critical regions in SIRT1 required for deacetylase activity as well as kinetic analyses of SIRT1 proteins.
