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Literature summary for 6.1.1.18 extracted from

  • Bullock, T.L.; Rodriguez-Hernandez, A.; Corigliano, E.M.; Perona, J.J.
    A rationally engineered misacylating aminoacyl-tRNA synthetase (2008), Proc. Natl. Acad. Sci. USA, 105, 7428-7433.
    View publication on PubMedView publication on EuropePMC
Search for more literature for 6.1.1.18

Cloned(Commentary)

Cloned (Comment) Organism
gene glnS, expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21 Escherichia coli

Crystallization (Commentary)

Crystallization (Comment) Organism
GlnRS-tRNAGln complex, 6.6 mg/ml protein in 10 mM PIPES, pH 7.5, 10 mM MgCl2, and 1.8-5.4 mM tRNA. The tRNA/analog solution is then mixed with equal volumes of a 6.3 mg/ml solution of GlnRS, containing 5mM PIPES, pH 7.0, and 5 mM 2-mercaptoethanol, X-ray diffraction structure determination and analysis at 2.6 A resolution Escherichia coli
purified recombinant GlnRS C229R-tRNAGln complex, a protein solution containing 6.3mg/ml GlnRS prepared in 5 mM PIPES, pH 7.0, 5 mM 2-mercaptoethanol, is mixed with the tRNAGln solution, X-ray diffraction structure determination and analysis at 2.6 A resolution Escherichia coli

Protein Variants

Protein Variants Comment Organism
C229R site-directed mutagenesis, transplanting the conserved arginine residue from glutamyl-tRNA synthetase, EC 6.1.1.17, to glutaminyltRNA synthetase improves the KM of GlnRS for noncognate glutamate Escherichia coli
C229R/Q255I site-directed mutagenesis, comparison of mutant activity with glutamate and glutamine to charge tRNAGln to the wild-type activity, the mutant shows no activity with L-Gln, but weakly with L-Glu Escherichia coli
C229R/Q255I/S227A/F233Y site-directed mutagenesis, comparison of mutant activity with glutamate and glutamine to charge tRNAGln to the wild-type activity, the mutant shows no activity with L-Gln, but activity with L-Glu Escherichia coli
additional information cumulative replacement of other primary binding site residues than Cys229 in GlnRS, with those of GluRS, only slightly improves the ability of the GlnRS active site to accommodate glutamate. Introduction of 22 amino acid replacements and one deletion, including substitution of the entire primary binding site and two surface loops adjacent to the region disrupted in C229R, improves the capacity of Escherichia coli GlnRS to synthesize misacylated Glu-tRNAGln by 16000fold. This hybrid enzyme recapitulates the function of misacylating GluRS enzymes found in organisms that synthesize Gln-tRNAGln by an alternative pathway, overview Escherichia coli
additional information the engineered mutant hybrid C229R Gln-RS, EC 6.1.1.18, shows activity with L-glutamine or L-glutamate and tRNAGln like the nondiscriminating enzyme, EC 6.1.1.24. Introduction of 22 amino acid replacements and one deletion, including substitution of the entire primary binding site and two surface loops adjacent to the region disrupted in the mutant C229R, improves the capacity of the mutant enzyme to synthesize misacylated Glu-tRNAGln by 16000fold, overview Escherichia coli
R30A site-directed mutagenesis, comparison of mutant activity with glutamate and glutamine to charge tRNAGln to the wild-type activity, the mutant shows no activity with L-Glu Escherichia coli
R30K site-directed mutagenesis, comparison of mutant activity with glutamate and glutamine to charge tRNAGln to the wild-type activity, the mutant shows weak activity with L-Glu Escherichia coli

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information kinetics of the mutant enzyme compared to wild-type GlnRS, EC 6.1.1.18, overview Escherichia coli
additional information
-
 additional information no KM value for L-glutamate with the wild-type enzyme due to no saturation, kinetics of wild-type and mutant enzymes, overview. Kinetics of extended-loop GlnRS mutants, overview Escherichia coli
0.21
-
 L-glutamine mutant C229R GlnRS, with tRNAGln Escherichia coli
0.26
-
 L-glutamine pH 7.5, 22°C, wild-type enzyme Escherichia coli
240
-
 L-glutamate mutant C229R GlnRS, with tRNAGln Escherichia coli

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+
-
 Escherichia coli

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
ATP + L-glutamate + tRNAGln Escherichia coli
-
 AMP + diphosphate + glutamyl-tRNAGln
-
 ?
ATP + L-glutamine + tRNAGln Escherichia coli
-
 AMP + diphosphate + L-glutaminyl-tRNAGln
-
 ?

Organism

Organism UniProt Comment Textmining
Escherichia coli
-
-
-
Escherichia coli P00962 gene glnS
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21 Escherichia coli

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
ATP + L-glutamate + tRNAGln primary binding pocket structure, overview Escherichia coli AMP + diphosphate + L-glutamyl-tRNAGln
-
 ?
ATP + L-glutamate + tRNAGln
-
 Escherichia coli AMP + diphosphate + glutamyl-tRNAGln
-
 ?
ATP + L-glutamate + tRNAGln activity also with Gln-RS, EC 6.1.1.18, mutant C229R Escherichia coli AMP + diphosphate + glutamyl-tRNAGln
-
 ?
ATP + L-glutamine + tRNAGln
-
 Escherichia coli AMP + diphosphate + L-glutaminyl-tRNAGln
-
 ?
ATP + L-glutamine + tRNAGln primary binding pocket structure, overview Escherichia coli AMP + diphosphate + L-glutaminyl-tRNAGln
-
 ?
additional information wild-type GlnRS catalyzes Glu-tRNAGln synthesis 1000000fold less efficiently than the cognate reaction Escherichia coli ?
-
 ?

Synonyms

Synonyms Comment Organism
GlnRS
-
 Escherichia coli
glutaminyltRNA synthetase
-
 Escherichia coli

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
22
-
 assay at room temperature Escherichia coli

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.00041
-
 L-glutamate mutant C229R GlnRS, with tRNAGln Escherichia coli
0.0025
-
 L-glutamine mutant C229R GlnRS, with tRNAGln Escherichia coli
0.046
-
 L-glutamate pH 7.5, 22°C, wild-type enzyme Escherichia coli
3.2
-
 L-glutamine pH 7.5, 22°C, wild-type enzyme Escherichia coli

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
 assay at Escherichia coli

Cofactor

Cofactor Comment Organism Structure
ATP
-
 Escherichia coli