Crystal structures reveal an elusive functional domain of pyrrolysyl-tRNA synthetase

Nat Chem Biol. 2017 Dec;13(12):1261-1266. doi: 10.1038/nchembio.2497. Epub 2017 Oct 16.


Pyrrolysyl-tRNA synthetase (PylRS) is a major tool in genetic code expansion using noncanonical amino acids, yet its structure and function are not completely understood. Here we describe the crystal structure of the previously uncharacterized essential N-terminal domain of this unique enzyme in complex with tRNAPyl. This structure explains why PylRS remains orthogonal in a broad range of organisms, from bacteria to humans. The structure also illustrates why tRNAPyl recognition by PylRS is anticodon independent: the anticodon does not contact the enzyme. Then, using standard microbiological culture equipment, we established a new method for laboratory evolution-a noncontinuous counterpart of the previously developed phage-assisted continuous evolution. With this method, we evolved novel PylRS variants with enhanced activity and amino acid specificity. Finally, we employed an evolved PylRS variant to determine its N-terminal domain structure and show how its mutations improve PylRS activity in the genetic encoding of a noncanonical amino acid.

MeSH terms

  • Amino Acyl-tRNA Synthetases / chemistry
  • Amino Acyl-tRNA Synthetases / genetics
  • Amino Acyl-tRNA Synthetases / metabolism*
  • Crystallography, X-Ray
  • Directed Molecular Evolution
  • Lysine / analogs & derivatives*
  • Lysine / chemistry
  • Lysine / metabolism
  • Methanosarcina / enzymology
  • Models, Molecular


  • Amino Acyl-tRNA Synthetases
  • pyrrolysine
  • Lysine

Associated data

  • PubChem-Substance/342584029
  • PubChem-Substance/342584033
  • PubChem-Substance/342584034
  • PubChem-Substance/342584035
  • PubChem-Substance/342584036
  • PubChem-Substance/342584037
  • PubChem-Substance/342584038
  • PubChem-Substance/342584039
  • PubChem-Substance/342584040
  • PubChem-Substance/342584030
  • PubChem-Substance/342584031
  • PubChem-Substance/342584032