An evolved pyrrolysyl-tRNA synthetase with polysubstrate specificity expands the toolbox for engineering enzymes with incorporation of noncanonical amino acids
Aminoacyl-tRNA synthetase (aaRS) is a core component for genetic code expansion (GCE), that enables the incorporation of noncanonical amino acids (ncAAs) into a protein. The aaRS with polyspecificity can be exploited in incorporating additional ncAAs into a protein without the evolution of new, orthogonal aaRS/tRNA pair. A variant (N346A/C348A) of pyrrolysyl-tRNA synthetase from Methanosarcina mazei (MmPylRS) exhibited a wide substrate scope of accepting over 40 phenylalanine derivatives. However, for most of the substrates, the incorporation efficiency was low. This study provides a PylRS variant with high activity and substrate promiscuity, which increases the utility of GCE in enzyme mechanism illustration and engineering.