@article{7080660d47bd409eb510e413b54a8c9a,
title = "Crystal structures reveal an elusive functional domain of pyrrolysyl-tRNA synthetase",
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.",
author = "Tateki Suzuki and Corwin Miller and Guo, {Li Tao} and Ho, {Joanne M.L.} and Bryson, {David I.} and Wang, {Yane Shih} and Liu, {David R.} and Dieter S{\"o}ll",
note = "Funding Information: The authors thank S. Melnikov and Y. Xiong (Yale University) for insightful discussions and intellectual contributions, A. Shinoda (Paul Scherrer Institute) and K. Yamashita (RIKEN) for advice on structure analysis, and S. Trauger (Small Molecule Mass Spectrometry Laboratory at Harvard University) for providing expertise with intact protein mass spectrometry analysis. This work was supported the US National Institutes of Health (NIH) R01EB022376 and R35GM118062 (to D.R.L.), and R01GM022854 and R35GM122560 (to D.S.), by the Defense Advanced Research Projects Agency N66001-12-C-4207 (to D.R.L.), by the Department of Energy DE-FG02-98ER20311 (to D.S.), and the Howard Hughes Medical Institute. D.I.B is supported by the National Institutes of Health under a Ruth L. Kirschstein National Research Service Award (F32GM106621). This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. Publisher Copyright: {\textcopyright} 2017 Nature America, Inc., part of Springer Nature. All rights reserved.",
year = "2017",
month = dec,
day = "1",
doi = "10.1038/nchembio.2497",
language = "English",
volume = "13",
pages = "1261--1266",
journal = "Nature Chemical Biology",
issn = "1552-4450",
publisher = "Nature Publishing Group",
number = "12",
}