title: Improved Incorporation of Noncanonical Amino Acids by an Engineered tRNA(Tyr) Suppressor.
abstract: The Methanocaldcoccus jannaschii tyrosyl-tRNA synthetase (TyrRS):tRNA(Tyr) cognate pair has been used to incorporate a large number of noncanonical amino acids (ncAAs) into recombinant proteins in Escherichia coli. However, the structural elements of the suppressor tRNA(Tyr) used in these experiments have not been examined for optimal performance. Here, we evaluate the steady-state kinetic parameters of wild-type M. jannaschii TyrRS and an evolved 3-nitrotyrosyl-tRNA synthetase (nitroTyrRS) toward several engineered tRNA(Tyr) suppressors, and we correlate aminoacylation properties with the efficiency and fidelity of superfolder green fluorescent protein (sfGFP) synthesis in vivo. Optimal ncAA-sfGFP synthesis correlates with improved aminoacylation kinetics for a tRNA(Tyr) amber suppressor with two substitutions in the anticodon loop (G34C/G37A), while four additional mutations in the D and variable loops, present in the tRNA(Tyr) used in all directed evolution experiments to date, are deleterious to function both in vivo and in vitro. These findings extend to three of four other evolved TyrRS enzymes that incorporate distinct ncAAs. Suppressor tRNAs elicit decreases in amino acid Km values for both TyrRS and nitroTyrRS, suggesting that direct anticodon recognition by TyrRS need not be an impediment to superior performance of this orthogonal system and offering insight into novel approaches for directed evolution. The G34C/G37A tRNA(Tyr) may enhance future incorporation of many ncAAs by engineered TyrRS enzymes.
authors.affiliation: Department of Chemistry, Portland State University , P.O. Box 751, Portland, Oregon 97207, United States.|Department of Biochemistry & Molecular Biology, Oregon Health & Sciences University , 3181 Southwest Sam Jackson Park Road, Portland, Oregon 97239, United States.
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Rauch
authors.affiliation: Department of Biochemistry and Biophysics, Oregon State University , 2011 Agriculture and Life Sciences Building, Corvallis, Oregon 97331, United States.
authors.forename: Joseph J
authors.initials: JJ
authors.lastname: Porter
authors.affiliation: Department of Biochemistry and Biophysics, Oregon State University , 2011 Agriculture and Life Sciences Building, Corvallis, Oregon 97331, United States.
authors.forename: Ryan A
authors.initials: RA
authors.lastname: Mehl
authors.affiliation: Department of Chemistry, Portland State University , P.O. Box 751, Portland, Oregon 97207, United States.|Department of Biochemistry & Molecular Biology, Oregon Health & Sciences University , 3181 Southwest Sam Jackson Park Road, Portland, Oregon 97239, United States.
authors.forename: John J
authors.initials: JJ
authors.lastname: Perona
mesh_terms: D000596:Amino Acids / Q000235:genetics*; D019843:Archaeal Proteins / Q000235:genetics* / Q000378:metabolism; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics; D007700:Kinetics; D063747:Methanocaldococcus / Q000201:enzymology*; D009154:Mutation; D009711:Nucleotides / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives / Q000235:genetics; D014448:Tyrosine-tRNA Ligase / Q000235:genetics* / Q000378:metabolism
chemical_list: D000596:Amino Acids; D019843:Archaeal Proteins; D009711:Nucleotides; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; C002744:3-nitrotyrosine; D014443:Tyrosine; D014448:Tyrosine-tRNA Ligase