title: An evolved aminoacyl-tRNA synthetase with atypical polysubstrate specificity.
abstract: We have employed a rapid fluorescence-based screen to assess the polyspecificity of several aminoacyl-tRNA synthetases (aaRSs) against an array of unnatural amino acids. We discovered that a p-cyanophenylalanine specific aminoacyl-tRNA synthetase (pCNF-RS) has high substrate permissivity for unnatural amino acids, while maintaining its ability to discriminate against the 20 canonical amino acids. This orthogonal pCNF-RS, together with its cognate amber nonsense suppressor tRNA, is able to selectively incorporate 18 unnatural amino acids into proteins, including trifluoroketone-, alkynyl-, and halogen-substituted amino acids. In an attempt to improve our understanding of this polyspecificity, the X-ray crystal structure of the aaRS-p-cyanophenylalanine complex was determined. A comparison of this structure with those of other mutant aaRSs showed that both binding site size and other more subtle features control substrate polyspecificity.
authors.affiliation: Department of Chemistry and Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States.
authors.forename: Douglas D
authors.initials: DD
authors.lastname: Young
authors.forename: Travis S
authors.initials: TS
authors.lastname: Young
authors.forename: Michael
authors.initials: M
authors.lastname: Jahnz
authors.forename: Insha
authors.initials: I
authors.lastname: Ahmad
authors.forename: Glen
authors.initials: G
authors.lastname: Spraggon
authors.forename: Peter G
authors.initials: PG
authors.lastname: Schultz
mesh_terms: D000409:Alanine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009570:Nitriles / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D012343:RNA, Transfer / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D010649:Phenylalanine / Q000031:analogs & derivatives
chemical_list: D000409:Alanine; D000604:Amino Acyl-tRNA Synthetases; D009570:Nitriles; D012343:RNA, Transfer; C530634:p-cyanophenylalanine; D010649:Phenylalanine