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

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

Author information

1
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA.
2
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA.
3
Howard Hughes Medical Institute, Cambridge, Massachusetts, USA.
4
Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA.
5
Department of Chemistry, Yale University, New Haven, Connecticut, USA.

Abstract

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.

PMID:
29035363
PMCID:
PMC5698177
DOI:
10.1038/nchembio.2497
[Indexed for MEDLINE]
Free PMC Article

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