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The  Methanosarcina mazei  pyrrolysyl-tRNA synthetase (PylRS)⋅tRNA Pyl  pair can be used to incorporate non-canonical amino acids (ncAAs) into proteins at installed amber stop codons. Although engineering of the PylRS active site generates diverse binding pockets, the substrate ranges are found similar in charging lysine and phenylalanine analogs. To expand the diversity of the ncAA side chains that can be incorporated  via  the PylRS⋅tRNA Pyl  pair, exploring remote interactions beyond the active site is an emerging approach in expanding the genetic code research. In this work, remote interactions between tRNA Pyl , the tRNA binding domain of PylRS, and/or an introduced non-structured linker between the N- and C-terminus of PylRS were studied. The substrate range of the PylRS⋅tRNA Pyl  pair was visualized by producing  sfGFP-UAG  gene products, which also indicated amber suppression efficiencies and substrate specificity. The unstructured loop linking the N-terminal and C-terminal domains (CTDs) of PylRS has been suggested to regulate the interaction between PylRS and tRNA Pyl . In exploring the detailed role of the loop region, different lengths of the linker were inserted into the junction between the N-terminal and the C-terminal domains of PylRS to unearth the impact on remote effects. Our findings suggest that the insertion of a moderate-length linker tunes the interface between PylRS and tRNA Pyl  and subsequently leads to improved suppression efficiencies. The suppression activity and the substrate specificity of PylRS were altered by introducing three mutations at or near the N-terminal domain of PylRS (N-PylRS). Using a N-PylRS⋅tRNA Pyl  pair, three ncAA substrates, two  S -benzyl cysteine and a histidine analog, were incorporated into the protein site specifically.

Linker and N-Terminal Domain Engineering of Pyrrolysyl-tRNA Synthetase for Substrate Range Shifting and Activity Enhancement