title: N-acetyl lysyl-tRNA synthetases evolved by a CcdB-based selection possess N-acetyl lysine specificity in vitro and in vivo.
abstract: Posttranslational modifications play a crucial role in modulating protein structure and function. Genetic incorporation of unnatural amino acids into a specific site of a protein facilitates the systematic study of protein modifications including acetylation. We here report the directed evolution of pyrrolysyl-tRNA synthetase (PylRS) from Methanosarcina mazei to create N-acetyl lysyl-tRNA synthetases (AcKRSs) using a new selection system based on the killing activity of the toxic ccdB gene product. The amino acid specificity of these and of published AckRSs was tested in vitro and in vivo, and the enzyme-kinetic properties of the AckRSs were evaluated for the first time.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.
authors.forename: Takuya
authors.initials: T
authors.lastname: Umehara
authors.forename: Jihyo
authors.initials: J
authors.lastname: Kim
authors.forename: Sangsik
authors.initials: S
authors.lastname: Lee
authors.forename: Li-Tao
authors.initials: LT
authors.lastname: Guo
authors.forename: Dieter
authors.initials: D
authors.lastname: Söll
authors.forename: Hee-Sung
authors.initials: HS
authors.lastname: Park
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019020:Directed Molecular Evolution; D008239:Lysine / Q000737:chemistry / Q000378:metabolism*; D008250:Lysine-tRNA Ligase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017020:Methanosarcina / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; C068356:CcdB protein, Plasmid F; D012346:RNA, Transfer, Amino Acyl; D008250:Lysine-tRNA Ligase; D008239:Lysine