Literature summary for 6.3.1.1 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 .

Cloned(Commentary)

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

Crystallization (Commentary)

Crystallization (Comment)	Organism
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

Crystallization (Comment)

Organism

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

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
0.6	-	L-aspartate	at pH 7.8 and 37°C	Leishmania donovani
1.2	-	L-aspartate	at pH 7.8 and 37°C	Leishmania donovani
5.95	-	NH3	at pH 7.8 and 37°C	Leishmania donovani
10.3	-	glutamine	at pH 7.8 and 37°C	Leishmania donovani

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining
cytoplasm	-	Leishmania donovani	5737	-

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	required	Trypanosoma brucei brucei

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
ATP + L-aspartate + NH3	Leishmania donovani	-	AMP + diphosphate + L-asparagine	-	?
ATP + L-aspartate + NH3	Leishmania donovani Bob	-	AMP + diphosphate + L-asparagine	-	?

Organism

Organism	UniProt	Comment	Textmining
Leishmania donovani	-	-	-
Leishmania donovani Bob	-	-	-
Trypanosoma brucei brucei	Q57WT9	-	-
Trypanosoma brucei brucei 927/4 GUTat10.1	Q57WT9	-	-

Purification (Commentary)

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

Source Tissue

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

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
ATP + L-aspartate + glutamine	-	Leishmania donovani	?	-	?
ATP + L-aspartate + glutamine	-	Leishmania donovani Bob	?	-	?
ATP + L-aspartate + NH3	-	Leishmania donovani	AMP + diphosphate + L-asparagine	-	?
ATP + L-aspartate + NH3	-	Leishmania donovani Bob	AMP + diphosphate + L-asparagine	-	?

Subunits

Subunits	Comment	Organism
?	x * 40000, calculated from amino acid sequence	Leishmania donovani
?	x * 40000, His6-tagged enzyme, SDS-PAGE	Leishmania donovani

Synonyms

Synonyms	Comment	Organism
AsnA	-	Leishmania donovani
AsnA	-	Trypanosoma brucei brucei
Asparagine synthetase A	-	Leishmania donovani
Asparagine synthetase A	-	Trypanosoma brucei brucei
Tb927.6.1880	-	Trypanosoma brucei brucei
TbASNA	-	Trypanosoma brucei brucei

Cofactor

Cofactor	Comment	Organism	Structure
ATP	-	Leishmania donovani
ATP	-	Trypanosoma brucei brucei

pI Value

Organism	Comment	pI Value Maximum	pI Value
Leishmania donovani	calculated from amino acid sequence	-	5.4

General Information

General Information	Comment	Organism
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
physiological function	the enzyme is essential for survival of the Leishmania parasite	Leishmania donovani