Suzuki, TatekiMiller, CorwinGuo, Li-TaoHo, Joanne M. L.Bryson, David I.Wang, Yane-ShihLiu, DavidSöll, Dieter2018-05-302017Suzuki, Tateki, Corwin Miller, Li-Tao Guo, Joanne M. L. Ho, David I. Bryson, Yane-Shih Wang, David R. Liu, and Dieter Söll. 2017. “Crystal structures reveal an elusive functional domain of pyrrolysyl-tRNA synthetase.” Nature chemical biology 13 (12): 1261-1266. doi:10.1038/nchembio.2497. http://dx.doi.org/10.1038/nchembio.2497.http://nrs.harvard.edu/urn-3:HUL.InstRepos:37068314Pyrrolysyl-tRNA synthetase (PylRS) is a major tool in genetic code expansion with non-canonical amino acids, yet understanding of its structure and activity is incomplete. 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. Using standard microbiological culture equipment, we then established a new method for laboratory evolution – a non-continuous counterpart of the previously developed phage-assisted continuous evolution. With this method, we evolved novel PylRS variants with enhanced activity and amino acid specificity. We finally 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 non-canonical amino acid.en-USJournal Article2018-05-3010.1038/nchembio.2497