Information on EC 6.3.5.6 - asparaginyl-tRNA synthase (glutamine-hydrolysing)

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The expected taxonomic range for this enzyme is: Archaea, Bacteria, Eukaryota

EC NUMBER
COMMENTARY hide
6.3.5.6
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RECOMMENDED NAME
GeneOntology No.
asparaginyl-tRNA synthase (glutamine-hydrolysing)
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + L-aspartyl-tRNAAsn + L-glutamine + H2O = ADP + phosphate + L-asparaginyl-tRNAAsn + L-glutamate
show the reaction diagram
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
transamidation
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-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Aminoacyl-tRNA biosynthesis
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L-asparagine biosynthesis III (tRNA-dependent)
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SYSTEMATIC NAME
IUBMB Comments
aspartyl-tRNAAsn:L-glutamine amido-ligase (ADP-forming)
This reaction forms part of a two-reaction system for producing asparaginyl-tRNA in Deinococcus radiodurans and other organisms lacking a specific enzyme for asparagine synthesis. In the first step, a non-discriminating ligase (EC 6.1.1.23, aspartate---tRNAAsn ligase) mischarges tRNAAsn with aspartate, leading to the formation of Asp-tRNAAsn. The aspartyl-tRNAAsn is not used in protein synthesis until the present enzyme converts it into asparaginyl-tRNAAsn (aspartyl-tRNAAsp is not a substrate for this reaction). Ammonia or asparagine can substitute for the preferred substrate glutamine.
CAS REGISTRY NUMBER
COMMENTARY hide
37211-76-0
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
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Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
no activity in Helicobacter pylori
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-
-
Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
Asp-tRNAAsn + ?
Asn-tRNAAsn + ?
show the reaction diagram
ATP + Asp-tRNA + L-Gln
?
show the reaction diagram
ATP + Asp-tRNAAsn + glutamine
ADP + phosphate + Asn-tRNAAsn + glutamate
show the reaction diagram
ATP + Asp-tRNAAsn + L-glutamine
ADP + phosphate + Asn-tRNAAsn + L-glutamate
show the reaction diagram
ATP + Asp-tRNAAsn + L-glutamine + H2O
ADP + phosphate + Asn-tRNAAsn + L-glutamate
show the reaction diagram
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-
-
-
?
ATP + Glu-tRNAGln + glutamine
ADP + Gln-tRNAGln + glutamate
show the reaction diagram
ATP + Glu-tRNAGln + glutamine
ADP + Gln-tRNAGln + glutamate + phosphate
show the reaction diagram
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the enzyme has no Glu-tRNAGln substrate in its host, but it can transamidate heterologous Glu-tRNAGln
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-
?
ATP + Glu-tRNAGln + L-glutamine
ADP + phosphate + Gln-tRNAGln + L-glutamate
show the reaction diagram
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the enzyme transamidates Asp-tRNAAsn and Glu-tRNAGln with similar efficiency. GatCAB uses the amide donor glutamine 129fold more efficiently than asparagine
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?
ATP + L-aspartyl-tRNAAsn + L-glutamine
ADP + phosphate + L-asparaginyl-tRNAAsn + L-glutamate
show the reaction diagram
Glu-tRNAGln + ?
Gln-tRNAGln + ?
show the reaction diagram
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
Asp-tRNAAsn + ?
Asn-tRNAAsn + ?
show the reaction diagram
ATP + Asp-tRNAAsn + L-glutamine + H2O
ADP + phosphate + Asn-tRNAAsn + L-glutamate
show the reaction diagram
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-
-
-
?
ATP + L-aspartyl-tRNAAsn + L-glutamine
ADP + phosphate + L-asparaginyl-tRNAAsn + L-glutamate
show the reaction diagram
Glu-tRNAGln + ?
Gln-tRNAGln + ?
show the reaction diagram
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(3Z)-3-[(2E,4E)-1-hydroxy-6-[(2S,4R)-2-(hydroxymethyl)-1,7-dimethyl-5-oxo-3,9,10-trioxatricyclo[4.3.1.02,4]dec-8-yl]-4-methylhepta-2,4-dien-1-ylidene]pyrrolidine-2,4-dione
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WS9326A
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little inhibitory activity at 0.1 mM
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WS9326C
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little inhibitory activity at 0.1 mM
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WS9326D
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specific inhibitor, moderate inhibition
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WS9326E
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moderate inhibition
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additional information
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not inhibited by N-ethylmaleimide, 5,5’-dithiobis(2-nitrobenzoic acid) and p-hydroxymercuribenzoate
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0224
Asn
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37°C, pH 7.2, amidotransferase activity
0.00095 - 0.0021
Asp-tRNAAsn
0.00118
Asp-tRNAGln
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37°C, pH 7.2, amidotransferase activity
0.2068
ATP
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37°C, pH 7.2, amidotransferase activity
0.0207 - 0.0509
Gln
additional information
additional information
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steady-state und pre-steady-state kinetics, association of dimeric DRS2, AdT, and tRNAAsn in a ternary complex, AdT binds tRNAAsn with a lower affinity than DRS2, overview
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.027
Asn
Helicobacter pylori
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37°C, pH 7.2, amidotransferase activity
0.25 - 1.3
Asp-tRNAAsn
3.61
Asp-tRNAGln
Helicobacter pylori
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37°C, pH 7.2, amidotransferase activity
6.1
ATP
Helicobacter pylori
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37°C, pH 7.2, amidotransferase activity
0.052 - 11.8
Gln
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.03
(3Z)-3-[(2E,4E)-1-hydroxy-6-[(2S,4R)-2-(hydroxymethyl)-1,7-dimethyl-5-oxo-3,9,10-trioxatricyclo[4.3.1.02,4]dec-8-yl]-4-methylhepta-2,4-dien-1-ylidene]pyrrolidine-2,4-dione
Brugia malayi
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pH and temperature not specified in the publication, selectively inhibits the Brugia malayi AsnRS and efficiently kills the adult Brugia malayi parasite
0.05
WS9326D
Brugia malayi
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pH and temperature not specified in the publication
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0.075
WS9326E
Brugia malayi
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pH and temperature not specified in the publication
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.2
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assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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primary
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
53600
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1 * 55000, GatA + 1 * 53600, GatB + 1* 11100, GatC, SDS-PAGE
55000
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1 * 55000, GatA + 1 * 53600, GatB + 1* 11100, GatC, SDS-PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
heterodimer
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Asp-AdT is active as a GatAB heterodimer
heterotrimer
additional information
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association of dimeric DRS2, AdT, and tRNAAsn in a ternary complex: a nondiscriminating aspartyl-tRNA synthetase, ND-DRS, first generates a mischarged aspartyl-tRNAAsn that dissociates from the enzyme and binds to a tRNA-dependent amidotransferase, AdT, which then converts the tRNA-bound aspartate into asparagine, the ND-DRS, tRNAAsn, and AdT assemble into a specific ribonucleoprotein complex called transamidosome that remains stable during the overall catalytic process, overview
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
structures of the catalytically active N-terminally truncated enzyme (residues 112-548) is solved by X-ray crystallography. The N-terminal domain contains a structured region with a novel fold featuring a lysine-rich helix that is shown by NMR to interact with tRNA. This is connected by an unstructured tether to the remainder of the enzyme, which is highly similar to the known structure of bacterial AsnRS
A8PWE4
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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thermophilic enzyme
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
32fold purification of recombinant enzyme, expressed in Escherichia coli
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DEAE-cellulose column chromatography and Ni-NTA column chromatography
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partial purification of recombinant enzyme, expressed in Escherichia coli
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purification of recombinant enzyme, expressed in Escherichia coli
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
canonical set of amidotransferase genes gatA, gatB and gatC is cloned and sequenced, the holoenzyme is overexpessed in Escherichia coli and exhibits both Asp-tRNAAsn and Glu-tRNAGln transamidation activities, the gatA, gatB and gatC genes are dispersed in the genome and encode polypeptides of 472, 470 and 90 amino acids, removal of the 44 carboxy-terminal amino acids of the GatA subunit only inhibits the Asp-AdT activity, but not the Glu-AdT activity
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cloning of the gatC, gatA and gatB genes, situated in an operon-like manner, encoding the GatCAB amidotransferase and overexpression in Escherichia coli BL21-Codon Plus-TM
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cloning of the gatCAB genes encoding the Asp/Glu-AdT and overexpression in Escherichia coli; presence of the gatCAB genes encoding Asp/Glu-AdT
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cloning of the gatCAB operon encoding the Asp-tRNAAsn amidotransferase and expression in Escherichia coli BL21-Codon Plus(DE3)-RIL
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expressed in Escherichia coli
A8PWE4
expression in Escherichia coli
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
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gatCAB genes encode Asp-AdT
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gatCAB genes encoding Glu/Asp-AdT are cloned and expressed in Escherichia coli
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overexpression in Escherichia coli
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presence of the gatCAB genes encoding Asp/Glu-AdT
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
DELTA 1-112
A8PWE4
structures of the catalytically active N-terminally truncated enzyme (residues 112-548) is solved by X-ray crystallography
S128T
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mutant protein retains significant glutaminase activity and transamidase activity in the presence of Gln
S152A
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mutant is glutaminase inactive
S152T
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mutant is glutaminase inactive
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
biotechnology
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inhibitor represents a lead scaffold to discover and develop antifilarial drugs
pharmacology
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enzyme may have potential as a species-specific therapeutic drug target
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