Cloned (Comment) | Organism |
---|---|
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 (Comment) | Organism |
---|---|
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 | Comment | Organism |
---|---|---|
A141W | site-directed mutagenesis, the mutant exhibits high tyrosine mischarging activity | Pyrococcus horikoshii |
D234A | site-directed mutagenesis, the mutant exhibits moderate tyrosine mischarging activity, the mutant PheRS incorrectly hydrolyze the cognate Phe-tRNAPhe | Pyrococcus horikoshii |
E127A | site-directed mutagenesis, the mutant exhibits low tyrosine mischarging activity | Pyrococcus horikoshii |
E219A | site-directed mutagenesis, the mutant is similar to the wild-type enzyme | Pyrococcus horikoshii |
F145A | site-directed mutagenesis, the mutant is similar to the wild-type enzyme | Pyrococcus horikoshii |
I216A | site-directed mutagenesis, the mutant is similar to the wild-type enzyme | Pyrococcus horikoshii |
L168A | site-directed mutagenesis, the mutant exhibits moderate tyrosine mischarging activity and shows reduced Tyr-tRNAPhe deacylation activity | Pyrococcus horikoshii |
L202A | site-directed mutagenesis, the mutant PheRS incorrectly hydrolyze the cognate Phe-tRNAPhe | Pyrococcus horikoshii |
L210A | site-directed mutagenesis, the mutant is similar to the wild-type enzyme | Pyrococcus horikoshii |
N217A | site-directed mutagenesis, the mutant exhibits high tyrosine mischarging activity and shows abolished Tyr-tRNAPhe deacylation activity | Pyrococcus horikoshii |
Q126A | site-directed mutagenesis, the mutant shows reduced Tyr-tRNAPhe deacylation activity | Pyrococcus horikoshii |
R137A | site-directed mutagenesis, the mutant exhibits low tyrosine mischarging activity and shows reduced Tyr-tRNAPhe deacylation activity | Pyrococcus horikoshii |
R223A | site-directed mutagenesis, the mutant exhibits moderate tyrosine mischarging activity and shows reduced Tyr-tRNAPhe deacylation activity | Pyrococcus horikoshii |
S211A | site-directed mutagenesis, the mutant PheRS incorrectly hydrolyze the cognate Phe-tRNAPhe | Pyrococcus horikoshii |
T221A | site-directed mutagenesis, the mutant is similar to the wild-type enzyme | Pyrococcus horikoshii |
T236A | site-directed mutagenesis, the mutant PheRS incorrectly hydrolyze the cognate Phe-tRNAPhe | Pyrococcus horikoshii |
Y189A | site-directed mutagenesis, the mutant exhibits low tyrosine mischarging activity | Pyrococcus horikoshii |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | - |
Pyrococcus horikoshii |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + L-phenylalanine + tRNAPhe | Pyrococcus horikoshii | - |
AMP + diphosphate + L-phenylalanyl-tRNAPhe | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Pyrococcus horikoshii | O73984 | - |
- |
Purification (Comment) | Organism |
---|---|
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 | Comment | Organism | Reaction ID |
---|---|---|---|
ATP + L-phenylalanine + tRNAPhe = AMP + diphosphate + L-phenylalanyl-tRNAPhe | residue N217 is essential for catalysis | Pyrococcus horikoshii |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + L-phenylalanine + tRNAPhe | - |
Pyrococcus horikoshii | AMP + diphosphate + L-phenylalanyl-tRNAPhe | - |
? | |
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 | Comment | Organism |
---|---|---|
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 | Comment | Organism |
---|---|---|
Phenylalanyl-tRNA synthetase | - |
Pyrococcus horikoshii |
PheRS | - |
Pyrococcus horikoshii |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
ATP | - |
Pyrococcus horikoshii |