Polyspecific pyrrolysyl-tRNA synthetases from directed evolution

Pyrrolysyl-tRNA synthetase (PyIRS) and its cognate tRNApyl have emerged as ideal translation components for genetic code innovation. Variants of the enzyme facilitate the incorporation > 100 noncanonical amino acids (ncAAs) into proteins. PyIRS variants were previously selected to acylate N⁶-acetyl-Lys (AcK) onto tRNApyl. Here, we examine an N⁶-acetyl-lysyl-tRNA synthetase (AcKRS), which is polyspecific (i.e., active with a broad range of ncAAs) and 30-fold more efficient with Phe derivatives than it is with AcK. Structural and biochemical data reveal the molecular basis of polyspecificity in AcKRS and in a PyIRS variant [iodo-phenylalanyl-tRNA synthetase (IFRS)] that displays both enhanced activity and substrate promiscuity over a chemical library of 313 ncAAs. IFRS, a product of directed evolution, has distinct binding modes for different ncAAs. These data indicate that in vivo selections do not produce optimally specific tRNA synthetases and suggest that translation fidelity will become an increasingly dominant factor in expanding the genetic code far beyond 20 amino acids.