Genetically encoding thio-acylated lysine derivatives for the study of sirtuin catalytic activity and specificity

Acylation is a posttranslational modification that involves the addition of an acyl group to the epsilon-nitrogen of lysine residues; for example, acetylation and propionylation. In recent years, thousands of lysine acylation sites have been identified on proteins involved in all cellular processes, but in human cells there are only 18 known deacetylases capable of hydrolyzing these acyl groups. Therefore, one of the most pressing questions in the field of lysine acylation is how 18 deacetylases recognize and differentiate between their different substrates. To facilitate the study of the interactions between NAD⁺-dependent sirtuin deacetylases and their substrates, we utilized genetic code expansion technology to genetically encode the co-translational incorporation of a thiolated version of propionyl lysine (TPrK). Based on studies of N^ε‐thioacetyl‐l‐lysine, the hydrolysis rate of TPrK is expected to be at least two orders of magnitude slower, compared to propionyl lysine. Hence, lysine thio-acylation is expected to function as a stable analogue of lysine acylation. We synthesized TPrK and identified an evolved tRNA synthetase for incorporation of TPrK into full-length proteins expressed living cells. Expression conditions in cultured mammalian cells were optimized and the deacetylation rate of TPrK by NAD⁺-dependent sirtuins was found to be slower than the deacetylation of propionyl lysine. The genetic encoding of thio-acyl lysine residues should enable the study of the sirtuin-specificity in living cells, by promoting the formation of a relatively stable complex between the enzymes and their substrates.