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Literature summary 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

Application

EC Number Application Comment Organism
6.3.5.4 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)

EC Number Cloned (Comment) Organism
6.3.1.1 expressed in Escherichia coli BL21 cells Leishmania donovani
6.3.1.1 gene Tb927.6.1880, recombinant expression of N-terminally His6-tagged enzyme in Escherichia coli strain BL21 Trypanosoma brucei brucei
6.3.5.4 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

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
6.3.1.1 purified recombinant His6-tagged apoenzyme, hanging drop vapor diffusion method, mixing of 0.001 ml of 73 mg/ml protein in 50 mM Tris-Cl, 200 mM NaCl, and 10 mM 2-mercaptoethanol, pH 7.5, with 0.001 ml of reservoir solution containing 0% w/v PEG 20000, 20% v/v PEG monomethyl ether 550, 0.03 M NPS (sodium nitrate, disodium hydrogen phosphate, ammonium sulfate), and 0.1 M MOPS/HEPES-Na, pH 7.5, 20°C, X-ray diffraction structure determination and analysis at 2.2 A resolution, modeling Trypanosoma brucei brucei

Protein Variants

EC Number Protein Variants Comment Organism
6.3.5.4 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]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
6.3.1.1 0.6
-
 L-aspartate at pH 7.8 and 37°C Leishmania donovani
6.3.1.1 1.2
-
 L-aspartate at pH 7.8 and 37°C Leishmania donovani
6.3.1.1 5.95
-
 NH3 at pH 7.8 and 37°C Leishmania donovani
6.3.1.1 10.3
-
 glutamine at pH 7.8 and 37°C Leishmania donovani
6.3.5.4 0.6
-
 L-aspartate recombinant enzyme, pH 7.8, 37°C Leishmania donovani
6.3.5.4 1.2
-
 ATP recombinant enzyme, pH 7.8, 37°C Leishmania donovani
6.3.5.4 5.95
-
 NH3 recombinant enzyme, pH 7.8, 37°C Leishmania donovani
6.3.5.4 10.3
-
 L-glutamine recombinant enzyme, pH 7.8, 37°C Leishmania donovani

Localization

EC Number Localization Comment Organism GeneOntology No. Textmining
6.3.1.1 cytoplasm
-
 Leishmania donovani 5737
-
6.3.5.4 cytosol
-
 Leishmania donovani 5829
-
6.3.5.4 mitochondrion
-
 Leishmania donovani 5739
-
6.3.5.4 additional information subcellular localization study using GFP-tagged enzyme Leishmania donovani
-
-

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
6.3.1.1 Mg2+ required Trypanosoma brucei brucei
6.3.5.4 Mg2+ required Leishmania donovani

Natural Substrates/ Products (Substrates)

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

Organism

EC Number Organism UniProt Comment Textmining
6.3.1.1 Leishmania donovani
-
-
-
6.3.1.1 Leishmania donovani Bob
-
-
-
6.3.1.1 Trypanosoma brucei brucei Q57WT9
-
-
6.3.1.1 Trypanosoma brucei brucei 927/4 GUTat10.1 Q57WT9
-
-
6.3.5.4 Leishmania donovani E9BLE1
-
-
6.3.5.4 Leishmania donovani BPK282A1 E9BLE1
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
6.3.1.1 recombinant His6-tagged enzyme from Escherichia coli strain BL21 by nickel affinity chromatography to over 95% purity, followed by gel filtration Trypanosoma brucei brucei
6.3.5.4 recombinant His6-tagged enzyme from Escherichia coli strain BL21 by nickel affinity chromatography to over 95% purity Leishmania donovani

Source Tissue

EC Number Source Tissue Comment Organism Textmining
6.3.1.1 kinetoplastid
-
 Trypanosoma brucei brucei
-
6.3.1.1 promastigote
-
 Leishmania donovani
-
6.3.5.4 kinetoplastid
-
 Leishmania donovani
-
6.3.5.4 promastigote
-
 Leishmania donovani
-

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
6.3.5.4 0.0696
-
 purified recombinant enzyme, pH 7.8, 37°C, substrate L-asparate Leishmania donovani

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
6.3.1.1 ATP + L-aspartate + glutamine
-
 Leishmania donovani ?
-
 ?
6.3.1.1 ATP + L-aspartate + glutamine
-
 Leishmania donovani Bob ?
-
 ?
6.3.1.1 ATP + L-aspartate + NH3
-
 Leishmania donovani AMP + diphosphate + L-asparagine
-
 ?
6.3.1.1 ATP + L-aspartate + NH3
-
 Leishmania donovani Bob AMP + diphosphate + L-asparagine
-
 ?
6.3.5.4 ATP + L-aspartate + L-glutamine + H2O
-
 Leishmania donovani AMP + diphosphate + L-asparagine + L-glutamate
-
 ?
6.3.5.4 ATP + L-aspartate + L-glutamine + H2O
-
 Leishmania donovani BPK282A1 AMP + diphosphate + L-asparagine + L-glutamate
-
 ?
6.3.5.4 ATP + L-aspartate + NH3 also reaction of EC 6.3.1.1 Leishmania donovani AMP + diphosphate + L-asparagine
-
 ?
6.3.5.4 ATP + L-aspartate + NH3 also reaction of EC 6.3.1.1 Leishmania donovani BPK282A1 AMP + diphosphate + L-asparagine
-
 ?
6.3.5.4 L-glutamine + H2O
-
 Leishmania donovani L-glutamate + NH3
-
 ?
6.3.5.4 L-glutamine + H2O
-
 Leishmania donovani BPK282A1 L-glutamate + NH3
-
 ?
6.3.5.4 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 ?
-
 ?
6.3.5.4 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

EC Number Subunits Comment Organism
6.3.1.1 ? x * 40000, calculated from amino acid sequence Leishmania donovani
6.3.1.1 ? x * 40000, His6-tagged enzyme, SDS-PAGE Leishmania donovani
6.3.5.4 ? x * 40000, about, sequence calculation, x * 44000, recombinant Hi6-tagged enzyme, SDS-PAGE Leishmania donovani

Synonyms

EC Number Synonyms Comment Organism
6.3.1.1 AsnA
-
 Leishmania donovani
6.3.1.1 AsnA
-
 Trypanosoma brucei brucei
6.3.1.1 Asparagine synthetase A
-
 Leishmania donovani
6.3.1.1 Asparagine synthetase A
-
 Trypanosoma brucei brucei
6.3.1.1 Tb927.6.1880
-
 Trypanosoma brucei brucei
6.3.1.1 TbASNA
-
 Trypanosoma brucei brucei
6.3.5.4 AsnA
-
 Leishmania donovani
6.3.5.4 Asparagine synthetase A
-
 Leishmania donovani
6.3.5.4 bacterial type asparagine synthetase A
-
 Leishmania donovani
6.3.5.4 LDBPK_300470
-
 Leishmania donovani

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
6.3.5.4 37
-
 assay at Leishmania donovani

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
6.3.5.4 7.8
-
 assay at Leishmania donovani

Cofactor

EC Number Cofactor Comment Organism Structure
6.3.1.1 ATP
-
 Leishmania donovani
6.3.1.1 ATP
-
 Trypanosoma brucei brucei
6.3.5.4 ATP
-
 Leishmania donovani

pI Value

EC Number Organism Comment pI Value Maximum pI Value
6.3.1.1 Leishmania donovani calculated from amino acid sequence
-
 5.4
6.3.5.4 Leishmania donovani sequence calculation
-
 5.4

General Information

EC Number General Information Comment Organism
6.3.1.1 evolution existence of a very significant conservation in active site residues between TbASNA and Escherichia coli AsnA. Asparagine synthetase is an enzyme that catalyzes the synthesis of asparagine from aspartate using ATP as the energy source in the presence of a nitrogen donor. The nitrogen donor can be glutamine or ammonia. Two families of asparagine synthetases are known to date: One is asparagine synthetase A (AsnA), which utilizes nitrogen only from an ammonia source reported from prokaryotes as well as from archaea. The other family is asparagine synthetase B (AsnB) whose members are found in prokaryotes and eukaryotes. Members of the AsnB family preferentially utilize glutamine as the nitrogen source, although they are capable of utilizing both glutamine and ammonia. Although they are eukaryotes, Leishmania and Trypanosoma surprisingly possess both ASNA and ASNB Trypanosoma brucei brucei
6.3.1.1 physiological function the enzyme is essential for survival of the Leishmania parasite Leishmania donovani
6.3.5.4 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
6.3.5.4 physiological function enzyme LdASNA is essential for survival of the Leishmania parasite Leishmania donovani