Expanding the Genetic Code with Lysine Aminoacylation

Lysine aminoacylation is a newly discovered protein post-translational modification that is found in humans. However, few studies have been implemented to further investigate its function, possibly due to limited tools to produce target proteins with homogeneously aminoacylated lysine residues at specific sites. To achieve this goal, we applied the genetic code expansion strategy, engineered pyrrolysyl-tRNA synthetase, and established orthogonal translation systems for ten types of lysine aminoacylation compatible for both bacterial and mammalian cells. Because metabolic enzymes are preferred substrate proteins of lysine aminoacylation, we tested the effect of lysine aminoacylation on metabolic enzymes and demonstrated that lysine valylation and tyrosylation impaired pyruvate kinase and glucose-6-phosphate dehydrogenase activities, respectively. Further <i>in vivo</i> studies showed that lysine valylation of pyruvate kinase decreased the basal glycolytic rate in living human cells. In summary, this work provides a toolbox to study lysine aminoacylation.