期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2014
卷号：111
期号：47
页码：16724-16729
DOI：10.1073/pnas.1419737111
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificancePyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNAPyl have emerged as ideal translation components for genetic code innovation. We found that a series of PylRS variants that were initially selected to be specific for the posttranslational modification N{varepsilon}-acetyl-L-Lys displayed polyspecificity [i.e., activity with a broad range of noncanonical amino acid (ncAA) substrates]. Our structural and biochemical data indicate that the engineered tRNA synthetases can accommodate ncAA substrates in multiple binding modes. The data further suggest that in vivo selections do not produce optimally specific tRNA synthetases and that translation fidelity will become an increasingly dominant factor in expanding the genetic code far beyond 20 amino acids. Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNAPyl have emerged as ideal translation components for genetic code innovation. Variants of the enzyme facilitate the incorporation >100 noncanonical amino acids (ncAAs) into proteins. PylRS variants were previously selected to acylate N{varepsilon}-acetyl-Lys (AcK) onto tRNAPyl. Here, we examine an N{varepsilon}-acetyl-lysyl-tRNA synthetase (AcKRS), which is polyspecific (i.e., active with a broad range of ncAAs) and 30-fold more efficient with Phe derivatives than it is with AcK. Structural and biochemical data reveal the molecular basis of polyspecificity in AcKRS and in a PylRS variant [iodo-phenylalanyl-tRNA synthetase (IFRS)] that displays both enhanced activity and substrate promiscuity over a chemical library of 313 ncAAs. IFRS, a product of directed evolution, has distinct binding modes for different ncAAs. These data indicate that in vivo selections do not produce optimally specific tRNA synthetases and suggest that translation fidelity will become an increasingly dominant factor in expanding the genetic code far beyond 20 amino acids.
