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Literature summary extracted from

  • Chuawong, P.; Hendrickson, T.L.
    The nondiscriminating aspartyl-tRNA synthetase from Helicobacter pylori: anticodon-binding domain mutations that impact tRNA specificity and heterologous toxicity (2006), Biochemistry, 45, 8079-8087.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
6.1.1.23 overexpression of His-tagged AsnRS in Escherichia coli strain DH5alpha, the enzyme is toxic when heterologously overexpressed in Escherichia coli, because of sequestration of tRNAAsn as Asp-tRNAAsn, this toxicity is rescued upon coexpression of the Helicobacter pylori Asp/Glu-Adt, EC 6.3.5.6 Helicobacter pylori
6.3.5.6 expression in Escherichia coli strain BL21(DE3), co-expression with the cytotoxic nondiscriminating aspartyl-tRNA synthetase, EC 6.1.1.23, which is toxic for the cells, rescues the toxicity, overview Helicobacter pylori

Protein Variants

EC Number Protein Variants Comment Organism
6.1.1.23 L81N site-directed mutagenesis, the mutation in the anticodon binding domain doubles the kcat for tRNAAsn as compared to the wild-type enzyme Helicobacter pylori
6.1.1.23 L81N/L86M site-directed mutagenesis, the mutation in the anticodon binding domain alters the tRNA specificity as compared to the wild-type enzyme, the L81N/L86M mutant does not follow Michaelis-Menten kinetics Helicobacter pylori
6.1.1.23 L86M site-directed mutagenesis, the mutation in the anticodon binding domain alters the tRNA specificity as compared to the wild-type enzyme Helicobacter pylori
6.1.1.23 additional information mutations in the anticodon binding domain of Helicobacter pylori ND-AspRS, e.g. at L81, L86, N82, and M87, reduce this enzyme’s ability to misacylate tRNAAsn and enhance tRNAAsp specificity, in a manner that correlates with the toxicity of the enzyme in Escherichia coli, overview Helicobacter pylori

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
6.1.1.23 additional information
-
 additional information the L81N/L86M mutant does not follow Michaelis-Menten kinetics Helicobacter pylori
6.1.1.23 0.77
-
 tRNAAsp pH 7.5, recombinant wild-type enzyme Helicobacter pylori
6.1.1.23 0.83
-
 tRNAAsn pH 7.5, recombinant wild-type enzyme Helicobacter pylori
6.1.1.23 0.85
-
 tRNAAsp pH 7.5, recombinant mutant L81N enzyme Helicobacter pylori
6.1.1.23 0.87
-
 tRNAAsp pH 7.5, recombinant mutant L86M enzyme Helicobacter pylori
6.1.1.23 1.87
-
 tRNAAsn pH 7.5, recombinant mutant L81N enzyme Helicobacter pylori
6.1.1.23 2.23
-
 tRNAAsn pH 7.5, recombinant mutant L86M enzyme Helicobacter pylori

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
6.1.1.23 Mg2+
-
 Helicobacter pylori
6.3.5.6 Mg2+
-
 Helicobacter pylori

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
6.1.1.23 ATP + L-aspartate + tRNAAsn Helicobacter pylori
-
 AMP + diphosphate + aspartyl-tRNAAsn
-
 ?
6.1.1.23 ATP + L-aspartate + tRNAAsp Helicobacter pylori
-
 AMP + diphosphate + aspartyl-tRNAAsp
-
 ?
6.1.1.23 additional information Helicobacter pylori in bacteria that lack AsnRS, AspRS is nondiscriminating and generates both Asp-tRNAAsp and the noncanonical, misacylated Asp-tRNAAsn, this misacylated tRNA is subsequently repaired by the glutamine-dependent Asp-tRNAAsn/Glu-tRNAGln amidotransferase, EC 6.3.5.6, increasing tRNAAsp specificity in an ND-AspRS diminishes in vivo toxicity ?
-
 ?
6.3.5.6 ATP + L-aspartyl-tRNAAsn + L-glutamine Helicobacter pylori
-
 ADP + phosphate + L-asparaginyl-tRNAAsn + L-glutamate
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
6.1.1.23 Helicobacter pylori
-
-
-
6.3.5.6 Helicobacter pylori
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
6.1.1.23 recombinant His-tagged AsnRS from Escherichia coli strain DH5alpha by nickel affinity chromatography Helicobacter pylori

Storage Stability

EC Number Storage Stability Organism
6.1.1.23 -20°C, purified recombinant enzyme, in 50% glycerol, and 33 mM phosphate, pH 7.4, 3 mM Tris-HCl, 1.5 mM 2-mercaptoethanol, and 0.5 mM phenylmethanesulfonyl fluoride, stable for months Helicobacter pylori

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
6.1.1.23 ATP + L-aspartate + tRNAAsn
-
 Helicobacter pylori AMP + diphosphate + aspartyl-tRNAAsn
-
 ?
6.1.1.23 ATP + L-aspartate + tRNAAsn recombinantly produced tRNA substrate Helicobacter pylori AMP + diphosphate + aspartyl-tRNAAsn
-
 ?
6.1.1.23 ATP + L-aspartate + tRNAAsp
-
 Helicobacter pylori AMP + diphosphate + aspartyl-tRNAAsp
-
 ?
6.1.1.23 ATP + L-aspartate + tRNAAsp recombinantly produced tRNA substrate Helicobacter pylori AMP + diphosphate + aspartyl-tRNAAsp
-
 ?
6.1.1.23 additional information in bacteria that lack AsnRS, AspRS is nondiscriminating and generates both Asp-tRNAAsp and the noncanonical, misacylated Asp-tRNAAsn, this misacylated tRNA is subsequently repaired by the glutamine-dependent Asp-tRNAAsn/Glu-tRNAGln amidotransferase, EC 6.3.5.6, increasing tRNAAsp specificity in an ND-AspRS diminishes in vivo toxicity Helicobacter pylori ?
-
 ?
6.1.1.23 additional information tRNA anticodon binding site structures, overview, Helicobacter pylori AspRS is a nondiscriminating enzyme that aminoacylates both tRNAAsp and tRNAAsn, ND-AspRS is 1.7times more efficient at aminoacylating tRNAAsp over tRNAAsn Helicobacter pylori ?
-
 ?
6.3.5.6 ATP + L-aspartyl-tRNAAsn + L-glutamine
-
 Helicobacter pylori ADP + phosphate + L-asparaginyl-tRNAAsn + L-glutamate
-
 ?
6.3.5.6 ATP + L-aspartyl-tRNAAsn + L-glutamine the Halobacter pylori Asp/Glu-Adt can utilize Escherichia coli Asp-tRNAAsn as a substrate, recombinantly produced tRNA substrate Helicobacter pylori ADP + phosphate + L-asparaginyl-tRNAAsn + L-glutamate
-
 ?
6.3.5.6 additional information in bacteria that lack AsnRS, EC 6.1.1.12, AspRS is nondiscriminating, EC 6.1.1.23, and generates both Asp-tRNAAsp and the noncanonical, misacylated Asp-tRNAAsn, this misacylated tRNA is subsequently repaired by the glutamine-dependent Asp-tRNAAsn/Glu-tRNAGln amidotransferase, overview Helicobacter pylori ?
-
 ?

Synonyms

EC Number Synonyms Comment Organism
6.1.1.23 ND-AspRS
-
 Helicobacter pylori
6.1.1.23 nondiscriminating aspartyl-tRNA synthetase
-
 Helicobacter pylori
6.3.5.6 Asp/Glu-Adt
-
 Helicobacter pylori
6.3.5.6 glutamine-dependent Asp-tRNAAsn/Glu-tRNAGln amidotransferase
-
 Helicobacter pylori

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
6.1.1.23 additional information
-
 additional information the wild-type enzyme has a kcat for tRNAAsp that is 60% higher than that of tRNAAsn Helicobacter pylori
6.1.1.23 0.014
-
 tRNAAsn pH 7.5, recombinant wild-type enzyme Helicobacter pylori
6.1.1.23 0.015
-
 tRNAAsn pH 7.5, recombinant mutant L86M enzyme Helicobacter pylori
6.1.1.23 0.021
-
 tRNAAsp pH 7.5, recombinant mutant L86M enzyme Helicobacter pylori
6.1.1.23 0.022
-
 tRNAAsp pH 7.5, recombinant wild-type enzyme Helicobacter pylori
6.1.1.23 0.026
-
 tRNAAsn pH 7.5, recombinant mutant L81N enzyme Helicobacter pylori
6.1.1.23 0.028
-
 tRNAAsp pH 7.5, recombinant mutant L81N enzyme Helicobacter pylori

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
6.1.1.23 7.5
-
 assay at Helicobacter pylori

Cofactor

EC Number Cofactor Comment Organism Structure
6.1.1.23 ATP
-
 Helicobacter pylori
6.3.5.6 ATP
-
 Helicobacter pylori