Structural Basis for Genetic-Code Expansion with Bulky Lysine Derivatives by an Engineered Pyrrolysyl-tRNA Synthetase

Cell Chem Biol. 2019 Jul 18;26(7):936-949.e13. doi: 10.1016/j.chembiol.2019.03.008. Epub 2019 Apr 25.


Pyrrolysyl-tRNA synthetase (PylRS) and tRNAPyl have been extensively used for genetic-code expansion. A Methanosarcina mazei PylRS mutant bearing the Y306A and Y384F mutations (PylRS(Y306A/Y384F)) encodes various bulky non-natural lysine derivatives by UAG. In this study, we examined how PylRS(Y306A/Y384F) recognizes many amino acids. Among 17 non-natural lysine derivatives, Nɛ-(benzyloxycarbonyl)lysine (ZLys) and 10 ortho/meta/para-substituted ZLys derivatives were efficiently ligated to tRNAPyl and were incorporated into proteins by PylRS(Y306A/Y384F). We determined crystal structures of 14 non-natural lysine derivatives bound to the PylRS(Y306A/Y384F) catalytic fragment. The meta- and para-substituted ZLys derivatives are snugly accommodated in the productive mode. In contrast, ZLys and the unsubstituted or ortho-substituted ZLys derivatives exhibited an alternative binding mode in addition to the productive mode. PylRS(Y306A/Y384F) displayed a high aminoacylation rate for ZLys, indicating that the double-binding mode minimally affects aminoacylation. These precise substrate recognition mechanisms by PylRS(Y306A/Y384F) may facilitate the structure-based design of novel non-natural amino acids.

Keywords: aminoacyl-tRNA synthetase; crystal structure; genetic code expansion; lysine derivatives; non-natural amino acid; pyrrolysyl-tRNA synthetase; specificity; structural basis; tRNA; translation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acyl-tRNA Synthetases / genetics
  • Amino Acyl-tRNA Synthetases / metabolism*
  • Crystallography, X-Ray
  • Escherichia coli
  • Genetic Code / genetics
  • Lysine / analogs & derivatives*
  • Lysine / chemistry
  • Lysine / genetics
  • Lysine / metabolism*
  • Methanosarcina / genetics
  • Models, Molecular
  • Protein Engineering / methods
  • RNA, Transfer / metabolism


  • N(epsilon)-(benzyloxycarbonyl)lysine
  • RNA, Transfer
  • Amino Acyl-tRNA Synthetases
  • pyrrolysine
  • Lysine