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Literature summary extracted from

  • Sasaki, H.M.; Sekine, S.; Sengoku, T.; Fukunaga, R.; Hattori, M.; Utsunomiya, Y.; Kuroishi, C.; Kuramitsu, S.; Shirouzu, M.; Yokoyama, S.
    Structural and mutational studies of the amino acid-editing domain from archaeal/eukaryal phenylalanyl-tRNA synthetase (2006), Proc. Natl. Acad. Sci. USA, 103, 14744-14749.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
6.1.1.20 expression of N-terminal fragment of the PheRS beta-subunit in Escherichia coli strain BL21(DE3), expression of selenomethionine-labeled N-terminal fragment of the PheRS beta-subunit in Escherichia coli strain B834(DE3) Pyrococcus horikoshii

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
6.1.1.20 purified recombinant selenomethionine-labeled N-terminal fragment of the PheRS beta-subunit, 0.001 ml of protein solution containing 36 mg/ml protein in 20 mM Tris-HCl buffer, pH 8.0, containing 200 mM NaCl, mixed with 0.001 ml of precipitant solution, containing 100 mM sodium citrate, pH 5.6, and 16.5% PEG 20000, covarage with a 1:1 mixture of paraffin oil and silicone, 20°C, 1 week, soaking in a soaked in a cryoprotectant solution containing 100 mM sodium citrate, pH 5.6, 18.2% PEG 20000, and 20% glycerol, X-ray diffraction structure determination and analysis at 1.94 A resolution Pyrococcus horikoshii

Protein Variants

EC Number Protein Variants Comment Organism
6.1.1.20 A141W site-directed mutagenesis, the mutant exhibits high tyrosine mischarging activity Pyrococcus horikoshii
6.1.1.20 D234A site-directed mutagenesis, the mutant exhibits moderate tyrosine mischarging activity, the mutant PheRS incorrectly hydrolyze the cognate Phe-tRNAPhe Pyrococcus horikoshii
6.1.1.20 E127A site-directed mutagenesis, the mutant exhibits low tyrosine mischarging activity Pyrococcus horikoshii
6.1.1.20 E219A site-directed mutagenesis, the mutant is similar to the wild-type enzyme Pyrococcus horikoshii
6.1.1.20 F145A site-directed mutagenesis, the mutant is similar to the wild-type enzyme Pyrococcus horikoshii
6.1.1.20 I216A site-directed mutagenesis, the mutant is similar to the wild-type enzyme Pyrococcus horikoshii
6.1.1.20 L168A site-directed mutagenesis, the mutant exhibits moderate tyrosine mischarging activity and shows reduced Tyr-tRNAPhe deacylation activity Pyrococcus horikoshii
6.1.1.20 L202A site-directed mutagenesis, the mutant PheRS incorrectly hydrolyze the cognate Phe-tRNAPhe Pyrococcus horikoshii
6.1.1.20 L210A site-directed mutagenesis, the mutant is similar to the wild-type enzyme Pyrococcus horikoshii
6.1.1.20 N217A site-directed mutagenesis, the mutant exhibits high tyrosine mischarging activity and shows abolished Tyr-tRNAPhe deacylation activity Pyrococcus horikoshii
6.1.1.20 Q126A site-directed mutagenesis, the mutant shows reduced Tyr-tRNAPhe deacylation activity Pyrococcus horikoshii
6.1.1.20 R137A site-directed mutagenesis, the mutant exhibits low tyrosine mischarging activity and shows reduced Tyr-tRNAPhe deacylation activity Pyrococcus horikoshii
6.1.1.20 R223A site-directed mutagenesis, the mutant exhibits moderate tyrosine mischarging activity and shows reduced Tyr-tRNAPhe deacylation activity Pyrococcus horikoshii
6.1.1.20 S211A site-directed mutagenesis, the mutant PheRS incorrectly hydrolyze the cognate Phe-tRNAPhe Pyrococcus horikoshii
6.1.1.20 T221A site-directed mutagenesis, the mutant is similar to the wild-type enzyme Pyrococcus horikoshii
6.1.1.20 T236A site-directed mutagenesis, the mutant PheRS incorrectly hydrolyze the cognate Phe-tRNAPhe Pyrococcus horikoshii
6.1.1.20 Y189A site-directed mutagenesis, the mutant exhibits low tyrosine mischarging activity Pyrococcus horikoshii

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
6.1.1.20 Mg2+
-
 Pyrococcus horikoshii

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
6.1.1.20 ATP + L-phenylalanine + tRNAPhe Pyrococcus horikoshii
-
 AMP + diphosphate + L-phenylalanyl-tRNAPhe
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
6.1.1.20 Pyrococcus horikoshii O73984
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
6.1.1.20 recombinant N-terminal fragment of the PheRS beta-subunit from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, recombinant selenomethionine-labeled N-terminal fragment of the PheRS beta-subunit from Escherichia coli strain B834(DE3) by anion exchange chromatography, adsorption chromatography, and gel filtration Pyrococcus horikoshii

Reaction

EC Number Reaction Comment Organism Reaction ID
6.1.1.20 ATP + L-phenylalanine + tRNAPhe = AMP + diphosphate + L-phenylalanyl-tRNAPhe residue N217 is essential for catalysis Pyrococcus horikoshii
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
6.1.1.20 ATP + L-phenylalanine + tRNAPhe
-
 Pyrococcus horikoshii AMP + diphosphate + L-phenylalanyl-tRNAPhe
-
 ?
6.1.1.20 ATP + L-phenylalanine + tRNAPhe editing mechanism of noncognate aminoacyl-tRNA involving domains B3 and B4 and residues Leu202, Ser211, Asp234, and Thr236, overview Pyrococcus horikoshii AMP + diphosphate + L-phenylalanyl-tRNAPhe
-
 ?

Subunits

EC Number Subunits Comment Organism
6.1.1.20 More the N-terminal fragment of the PheRS beta-subunit includes the editing domain B3/4, which has archaea/eukarya-specific insertions/deletions and adopts a different orientation relative to other domains, as compared with that of bacterial PheRS, structure, overview Pyrococcus horikoshii

Synonyms

EC Number Synonyms Comment Organism
6.1.1.20 Phenylalanyl-tRNA synthetase
-
 Pyrococcus horikoshii
6.1.1.20 PheRS
-
 Pyrococcus horikoshii

Cofactor

EC Number Cofactor Comment Organism Structure
6.1.1.20 ATP
-
 Pyrococcus horikoshii