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Literature summary for 6.3.5.4 extracted from

  • Faria, J.; Loureiro, I.; Santarem, N.; Macedo-Ribeiro, S.; Tavares, J.; Cordeiro-da-Silva, A.
    Leishmania infantum asparagine synthetase A is dispensable for parasites survival and infectivity (2016), PLoS Negl. Trop. Dis., 10, e0004365 .
    View publication on PubMedView publication on EuropePMC
Search for more literature for 6.3.5.4

Application

Application Comment Organism
drug development enzyme AS-A is unlikely to be a suitable drug target against Leishmania parasites Leishmania infantum

Cloned(Commentary)

Cloned (Comment) Organism
DNA and amino acid sequence determination and analysis, sequence comparisons, recombinant expression of N-terminally His6-tagged enzyme in Escherichia coli strain BL21(DE3), enzyme expression analysis Leishmania infantum

Protein Variants

Protein Variants Comment Organism
additional information generation of a AS-A gene knockout mutant by targeted gene replacement Leishmania infantum

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information steady-state kinetics Leishmania infantum
1.12
-
 NH3 pH 7.6, 37°C, recombinant enzyme Leishmania infantum
1.47
-
 ATP pH 7.6, 37°C, recombinant enzyme Leishmania infantum
1.71
-
 L-glutamine pH 7.6, 37°C, recombinant enzyme Leishmania infantum
6.21
-
 L-aspartate pH 7.6, 37°C, recombinant enzyme Leishmania infantum

Localization

Localization Comment Organism GeneOntology No. Textmining
cytosol
-
 Leishmania infantum 5829
-
additional information enzyme subcellular localisation determination, no localization of the Leishmania infantum enzyme in mitochondria Leishmania infantum
-
-

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
78800
-
 recombinant enzyme, gel filtration Leishmania infantum

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
ATP + L-aspartate + L-glutamine + H2O Leishmania infantum
-
 AMP + diphosphate + L-asparagine + L-glutamate
-
 ?
ATP + L-aspartate + L-glutamine + H2O Leishmania infantum JPCM5
-
 AMP + diphosphate + L-asparagine + L-glutamate
-
 ?
ATP + L-aspartate + NH3 Leishmania infantum
-
 AMP + diphosphate + L-asparagine
-
 ?
ATP + L-aspartate + NH3 Leishmania infantum JPCM5
-
 AMP + diphosphate + L-asparagine
-
 ?

Organism

Organism UniProt Comment Textmining
Leishmania infantum A4I213
-
-
Leishmania infantum JPCM5 A4I213
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant N-terminally His6-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, gel filtration, and ion exchange chromatography Leishmania infantum

Reaction

Reaction Comment Organism Reaction ID
ATP + L-aspartate + L-glutamine + H2O = AMP + diphosphate + L-asparagine + L-glutamate the enzyme forms a crucial beta-aspartyl-AMP-Mg2+ intermediate, which then undergoes a nucleophilic attack of ammonia, forming Asn and releasing AMP and diphosphate. Ammonia can be free or glutamine-derived Leishmania infantum
a

Source Tissue

Source Tissue Comment Organism Textmining
promastigote enzyme expression analysis Leishmania infantum
-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
ATP + L-aspartate + L-glutamine + H2O
-
 Leishmania infantum AMP + diphosphate + L-asparagine + L-glutamate
-
 ?
ATP + L-aspartate + L-glutamine + H2O
-
 Leishmania infantum JPCM5 AMP + diphosphate + L-asparagine + L-glutamate
-
 ?
ATP + L-aspartate + NH3
-
 Leishmania infantum AMP + diphosphate + L-asparagine
-
 ?
ATP + L-aspartate + NH3
-
 Leishmania infantum JPCM5 AMP + diphosphate + L-asparagine
-
 ?

Subunits

Subunits Comment Organism
homodimer 2 * 42000, recombinant enzyme, SDS-PAGE, 2 * 39800, native enzyme, SDS-PAGE Leishmania infantum

Synonyms

Synonyms Comment Organism
AS-A
-
 Leishmania infantum
Asparagine synthetase
-
 Leishmania infantum
Asparagine synthetase A
-
 Leishmania infantum
LiAS-A
-
 Leishmania infantum
More cf. EC 6.3.1.1 Leishmania infantum

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
37
-
 assay at Leishmania infantum

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
4.18
-
 NH3 pH 7.6, 37°C, recombinant enzyme Leishmania infantum
4.51
-
 L-glutamine pH 7.6, 37°C, recombinant enzyme Leishmania infantum
7.46
-
 ATP pH 7.6, 37°C, recombinant enzyme Leishmania infantum
9.19
-
 L-aspartate pH 7.6, 37°C, recombinant enzyme Leishmania infantum

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.6
-
 assay at Leishmania infantum

Cofactor

Cofactor Comment Organism Structure
ATP
-
 Leishmania infantum

General Information

General Information Comment Organism
malfunction enzyme AS-A ablation drives parasites auxotrophic to asparagine, but LiAS-A is not detrimental for parasite survival, growth or infectivity. The parasite burden in the spleen and liver of female BALB/c mice is not statistically different in LiASA mutants when compared to the wild-type Leishmania infantum
metabolism asparagine synthetase (AS) is responsible for the conversion of aspartate into Asn in an ATP-dependent manner, using ammonia or glutamine as a nitrogen source. There are two structurally distinct AS: the strictly ammonia-dependent type A, and the type B, which preferably uses glutamine Leishmania infantum
additional information enzyme structure homology modeling, structure and sequence comparisons of the enzymes from Leishmania infantum and Leishmania major, overview Leishmania infantum
physiological function Leishmania infantum encodes for a functional AS-A enzyme, which uses either ammonia or glutamine as nitrogen donor for asparagine synthesis. LiAS-A is required for promastigotes growth only in asparagine limiting conditions Leishmania infantum

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.48
-
 ATP pH 7.6, 37°C, recombinant enzyme Leishmania infantum
2.64
-
 L-glutamine pH 7.6, 37°C, recombinant enzyme Leishmania infantum
2.84
-
 L-aspartate pH 7.6, 37°C, recombinant enzyme Leishmania infantum
6.66
-
 NH3 pH 7.6, 37°C, recombinant enzyme Leishmania infantum