Pyrrolysine (Pyl), the 22nd natural amino acid, is genetically encoded by UAG and inserted into proteins by the unique suppressor tRNAPyl (ref. 1). The Methanosarcinaceae produce Pyl and express Pyl-containing methyltransferases that allow growth on methylamines2. Homologous methyltransferases and the Pyl biosynthetic and coding machinery are also found in two bacterial species1,3. Pyl coding is maintained by pyrrolysyl-tRNA synthetase (PylRS), which catalyses the formation of Pyl-tRNAPyl (refs 4, 5). Pyl is not a recent addition to the genetic code. PylRS was already present in the last universal common ancestor6; it then persisted in organisms that utilize methylamines as energy sources. Recent protein engineering efforts added non-canonical amino acids to the genetic code7,8. This technology relies on the directed evolution of an ‘orthogonal’ tRNA synthetase–tRNA pair in which an engineered aminoacyl-tRNA synthetase (aaRS) specifically and exclusively acylates the orthogonal tRNA with a non-canonical amino acid. For Pyl the natural evolutionary process developed such a system some 3 billion years ago. When transformed into Escherichia coli, Methanosarcina barkeri PylRS and tRNAPyl function as an orthogonal pair in vivo5,9. Here we show that Desulfitobacterium hafniense PylRS–tRNAPyl is an orthogonal pair in vitro and in vivo, and present the crystal structure of this orthogonal pair. The ancient emergence of PylRS–tRNAPyl allowed the evolution of unique structural features in both the protein and the tRNA. These structural elements manifest an intricate, specialized aaRS–tRNA interaction surface that is highly distinct from those observed in any other known aaRS–tRNA complex; it is this general property that underlies the molecular basis of orthogonality.
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We thank the beamline staff at BL41XU of SPring-8 (Harima, Japan) and NW12 of PF-AR (Tsukuba, Japan) for technical help during data collection. P.O. holds a National Science Foundation postdoctoral fellowship in Biological Informatics. This work was supported by grants from the Japan Science and Technology Agency (to O.N.), from the National Project on Protein Structural and Functional Analyses of the Ministry of Education, Culture, Sports, Science and Technology (to O.N.), from the Ministry of Education, Culture, Sports, Science and Technology (to R.I. and O.N.), from the Mitsubishi Foundation (to O.N.), from the Kurata Memorial Hitachi Science and Technology Foundation (to O.N.), from the National Institute of General Medical Sciences (to D.S.), from the Department of Energy (to D.S.), and from the National Science Foundation (to D.S.).
Author Contributions K.N. performed purification, crystallization and structure determination. S.G. and T.U. conducted biochemical analyses. R.I. performed molecular dynamics. Y.A., R.I. and O.N. assisted the structure determination. P.O’D. analysed the data and performed bioinformatic analysis. P.O’D., K.N., O.N. and D.S. wrote the paper. O.N. and D.S. conceived and supervised the work.
This file contains a Supplementary Discussion, Supplementary References, Supplementary Figures 1-7 and Supplementary Tables 1-2
About this article
Nature Reviews Microbiology (2015)