Probing the Active Site of Deubiquitinase USP30 with Noncanonical Tryptophan Analogues.

pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA have been evolved to generate genetically encoded noncanonical amino acids (ncAAs). Use of tryptophan (Trp) analogues with pyrrole ring modification for their spatial and polarity tuning in enzyme activity and substrate specificity is still limited. Herein, we report the application of an evolved PylRS, FOWRS2, for efficient incorporation of five Trp analogues into the deubiquitinase USP30 to decipher the role of W475 for diubiquitin selectivity.

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

The pyrrolysyl-tRNA synthetase (PylRS)⋅tRNA 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 the PylRS⋅tRNA pair, exploring remote interactions beyond the active site is an emerging approach in expanding the genetic code research.