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Literature summary for 6.3.5.4 extracted from

  • Manhas, R.; Tripathi, P.; Khan, S.; Sethu Lakshmi, B.; Lal, S.K.; Gowri, V.S.; Sharma, A.; Madhubala, R.
    Identification and functional characterization of a novel bacterial type asparagine synthetase A a tRNA synthetase paralog from Leishmania donovani (2014), J. Biol. Chem., 289, 12096-12108 .
    View publication on PubMedView publication on EuropePMC
Search for more literature for 6.3.5.4

Application

Application Comment Organism
drug development the absence of an LdASNA homologue from humans and its essentiality for the parasites make LdASNA a drug target Leishmania donovani

Cloned(Commentary)

Cloned (Comment) Organism
gene LDBPK_300470, DNA and amino acid sequence determination and analysis, sequence comaparisons and phylogenetic analysis, cloning of the pSP-alpha-blast-alpha-ASNA and pSP72-alpha-neo-alpha-GFP-ASNA episome, overexpression of GFP-tagged ASNA in Leishmania donovani Bob strain MHOM/SD/62/1SCL2D, recombinant expression of N-terminally His6-tagged enzyme in Escherichia coli strain BL21 Leishmania donovani

Protein Variants

Protein Variants Comment Organism
additional information gene deletion mutations of gene asnA are attempted via targeted gene replacement. Gene deletion of LdASNA leads to growth delay in mutants. Chromosomal null mutants of LdASNA cannot be obtained as the double transfectant mutants show aneuploidy Leishmania donovani

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.6
-
 L-aspartate recombinant enzyme, pH 7.8, 37°C Leishmania donovani
1.2
-
 ATP recombinant enzyme, pH 7.8, 37°C Leishmania donovani
5.95
-
 NH3 recombinant enzyme, pH 7.8, 37°C Leishmania donovani
10.3
-
 L-glutamine recombinant enzyme, pH 7.8, 37°C Leishmania donovani

Localization

Localization Comment Organism GeneOntology No. Textmining
cytosol
-
 Leishmania donovani 5829
-
mitochondrion
-
 Leishmania donovani 5739
-
additional information subcellular localization study using GFP-tagged enzyme Leishmania donovani
-
-

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ required Leishmania donovani

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
ATP + L-aspartate + L-glutamine + H2O Leishmania donovani
-
 AMP + diphosphate + L-asparagine + L-glutamate
-
 ?
ATP + L-aspartate + L-glutamine + H2O Leishmania donovani BPK282A1
-
 AMP + diphosphate + L-asparagine + L-glutamate
-
 ?
ATP + L-aspartate + NH3 Leishmania donovani also reaction of EC 6.3.1.1 AMP + diphosphate + L-asparagine
-
 ?
ATP + L-aspartate + NH3 Leishmania donovani BPK282A1 also reaction of EC 6.3.1.1 AMP + diphosphate + L-asparagine
-
 ?
L-glutamine + H2O Leishmania donovani
-
 L-glutamate + NH3
-
 ?
L-glutamine + H2O Leishmania donovani BPK282A1
-
 L-glutamate + NH3
-
 ?

Organism

Organism UniProt Comment Textmining
Leishmania donovani E9BLE1
-
-
Leishmania donovani BPK282A1 E9BLE1
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His6-tagged enzyme from Escherichia coli strain BL21 by nickel affinity chromatography to over 95% purity Leishmania donovani

Source Tissue

Source Tissue Comment Organism Textmining
kinetoplastid
-
 Leishmania donovani
-
promastigote
-
 Leishmania donovani
-

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
0.0696
-
 purified recombinant enzyme, pH 7.8, 37°C, substrate L-asparate Leishmania donovani

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
ATP + L-aspartate + L-glutamine + H2O
-
 Leishmania donovani AMP + diphosphate + L-asparagine + L-glutamate
-
 ?
ATP + L-aspartate + L-glutamine + H2O
-
 Leishmania donovani BPK282A1 AMP + diphosphate + L-asparagine + L-glutamate
-
 ?
ATP + L-aspartate + NH3 also reaction of EC 6.3.1.1 Leishmania donovani AMP + diphosphate + L-asparagine
-
 ?
ATP + L-aspartate + NH3 also reaction of EC 6.3.1.1 Leishmania donovani BPK282A1 AMP + diphosphate + L-asparagine
-
 ?
L-glutamine + H2O
-
 Leishmania donovani L-glutamate + NH3
-
 ?
L-glutamine + H2O
-
 Leishmania donovani BPK282A1 L-glutamate + NH3
-
 ?
additional information asparagine is formed in two steps: the beta-carboxylate group of aspartate is first activated by ATP to form an aminoacyl-AMP before its amidation by a nucleophilic attack with an ammonium ion. LdASNA is active and preferentially utilizes ammonia, although it is also capable of utilizing glutamine as a nitrogen source Leishmania donovani ?
-
 ?
additional information asparagine is formed in two steps: the beta-carboxylate group of aspartate is first activated by ATP to form an aminoacyl-AMP before its amidation by a nucleophilic attack with an ammonium ion. LdASNA is active and preferentially utilizes ammonia, although it is also capable of utilizing glutamine as a nitrogen source Leishmania donovani BPK282A1 ?
-
 ?

Subunits

Subunits Comment Organism
? x * 40000, about, sequence calculation, x * 44000, recombinant Hi6-tagged enzyme, SDS-PAGE Leishmania donovani

Synonyms

Synonyms Comment Organism
AsnA
-
 Leishmania donovani
Asparagine synthetase A
-
 Leishmania donovani
bacterial type asparagine synthetase A
-
 Leishmania donovani
LDBPK_300470
-
 Leishmania donovani

Temperature Optimum [°C]

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

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.8
-
 assay at Leishmania donovani

Cofactor

Cofactor Comment Organism Structure
ATP
-
 Leishmania donovani

pI Value

Organism Comment pI Value Maximum pI Value
Leishmania donovani sequence calculation
-
 5.4

General Information

General Information Comment Organism
metabolism asparagine is formed by two structurally distinct asparagine synthetases in prokaryotes. One is the ammonia-utilizing asparagine synthetase A (AsnA, EC 6.3.1.1), and the other is asparagine synthetase B (AsnB, EC 6.3.5.4) that uses glutamine or ammonia as a nitrogen source. Sequence-based analysis suggests that Leishmania spp. possess the asparagine tRNA synthetase paralogue asparagine synthetase A (LdASNA) that is ammonia-dependent, but enzyme LdASNA from Leishmania donovani is both ammonia- and glutamine-dependent, EC 6.3.5.4 Leishmania donovani
physiological function enzyme LdASNA is essential for survival of the Leishmania parasite Leishmania donovani