Crystal Structures Reveal an Elusive Functional Domain of Pyrrolysyl-Trna Synthetase
Nature chemical biology(2017)
摘要
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 tRNA(Pyl). This structure explains why PylRS remains orthogonal in a broad range of organisms, from bacteria to humans. The structure also illustrates why tRNA(Pyl) 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.
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关键词
Protein design,Proteins,X-ray crystallography,Chemistry/Food Science,general,Biochemical Engineering,Biochemistry,Cell Biology,Bioorganic Chemistry
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