Information on EC 6.1.1.23 - aspartate-tRNAAsn ligase

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

EC NUMBER
COMMENTARY
6.1.1.23
-
RECOMMENDED NAME
GeneOntology No.
aspartate-tRNAAsn ligase
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
ATP + L-aspartate + tRNAAsx = AMP + diphosphate + aspartyl-tRNAAsx
show the reaction diagram
-
-
-
-
ATP + L-aspartate + tRNAAsx = AMP + diphosphate + aspartyl-tRNAAsx
show the reaction diagram
residues H31 and G83 are important determinants for substrate specificity towards tRNA substrates, molecular modeling
-
ATP + L-aspartate + tRNAAsx = AMP + diphosphate + aspartyl-tRNAAsx
show the reaction diagram
substrate anticodon, GUC for aspartate and GUU for asparagine, recognition site structure and mechanism
-
ATP + L-aspartate + tRNAAsx = AMP + diphosphate + aspartyl-tRNAAsx
show the reaction diagram
via reaction intermediate L-aspartyl adenylate
-
REACTION TYPE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
Aminoacylation
-
-
-
-
Aminoacylation
-
-
esterification
-
-
-
-
PATHWAY
KEGG Link
MetaCyc Link
Aminoacyl-tRNA biosynthesis
-
SYSTEMATIC NAME
IUBMB Comments
L-aspartate:tRNAAsx ligase (AMP-forming)
When this enzyme acts on tRNAAsp, it catalyses the same reaction as EC 6.1.1.12, aspartate---tRNA ligase. It has, however, diminished discrimination, so that it can also form aspartyl-tRNAAsn. This relaxation of specificity has been found to result from the absence of a loop in the tRNA that specifically recognizes the third position of the anticodon [1]. This accounts for the ability of this enzyme in, for example, Thermus thermophilus, to recognize both tRNAAsp (GUC anticodon) and tRNAAsn (GUU anticodon). The aspartyl-tRNAAsn is not used in protein synthesis until it is converted by EC 6.3.5.6, asparaginyl-tRNA synthase (glutamine-hydrolysing), into asparaginyl-tRNAAsn.
SYNONYMS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
aspartate tRNA synthetase
-
-
-
-
Aspartic acid translase
-
-
-
-
Aspartyl ribonucleic synthetase
-
-
-
-
aspartyl tRNA synthetase
-
-
-
-
aspartyl-transfer ribonucleate synthetase
-
-
-
-
Aspartyl-transfer ribonucleic acid synthetase
-
-
-
-
Aspartyl-transfer RNA synthetase
-
-
-
-
Aspartyl-tRNA synthetase
C4LZN0
-
Aspartyl-tRNA synthetase
-
-
Aspartyl-tRNA synthetase
-
-
Aspartyl-tRNA synthetase
-
-
Aspartyl-tRNA synthetase
-
-
AspRS
C4LZN0
-
ND-AspRS
O07683
-
ND-AspRS
Halobacterium salinarum NRC-1
O07683
-
-
non-discriminating aspartyl-tRNA synthetase
O07683
-
non-discriminating aspartyl-tRNA synthetase
Halobacterium salinarum NRC-1
O07683
-
-
non-discriminating aspartyl-tRNA synthetase
-
-
nondicriminating AspRS
-
-
nondiscriminating aspartyl-tRNA synthetase
-
-
-
-
nondiscriminating aspartyl-tRNA synthetase
-
-
nondiscriminating aspartyl-tRNA synthetase
-
-
nondiscriminating aspartyl-tRNA synthetase
O26328
-
nondiscriminating aspartyl-tRNA synthetase
Methanothermobacter thermautotrophicus DSM 1053
O26328
-
-
nondiscriminating aspartyl-tRNA synthetase
-
-
nondiscriminating aspartyl-tRNA synthetase
-
-
nondiscriminating aspartyl-tRNA synthetase
-
-
nondiscriminating AspRS
-
-
Synthetase, aspartyl-transfer ribonucleate
-
-
-
-
EC 6.1.1.12
-
-
related
-
additional information
C4LZN0
AspRS is a class IIb aminoacyl-tRNA synthetase
CAS REGISTRY NUMBER
COMMENTARY
9027-32-1
-
ORGANISM
COMMENTARY
LITERATURE
SEQUENCE CODE
SEQUENCE DB
SOURCE
gene aspS, ND-AspRS; strain NRC-1, gene aspS
SwissProt
Manually annotated by BRENDA team
Halobacterium salinarum NRC-1
gene aspS, ND-AspRS; strain NRC-1, gene aspS
SwissProt
Manually annotated by BRENDA team
gene DARS2
-
-
Manually annotated by BRENDA team
Methanothermobacter thermautotrophicus DSM 1053
-
UniProt
Manually annotated by BRENDA team
no activity in Sulfolobus tokodaii
-
-
-
Manually annotated by BRENDA team
no activity in Sulfolobus tokodaii 7
-
-
-
Manually annotated by BRENDA team
; gene aspS
-
-
Manually annotated by BRENDA team
strain PAO1
-
-
Manually annotated by BRENDA team
a crenarchaeon, strain 7
-
-
Manually annotated by BRENDA team
a crenarchaeon, strain 7, gene ST0205
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
physiological function
-
during tRNA-dependent asparagine formation, tRNAAsn promotes assembly of a ribonucleoprotein particle called transamidosome that allows channelling of the aa-tRNA from non-discriminating aspartyl-tRNA synthetase active site to the GatCAB amidotransferase site. A transamidosome particle is formed by two GatCABs, two dimeric nondiscriminating-AspRSs and four tRNAsAsn molecules. In the complex, only two tRNAs are bound in a functional state, whereas the two other ones act as an RNA scaffold enabling release of the asparaginyl-tRNAAsn without dissociation of the complex. The transamidosome constitutes a transfer-ribonucleoprotein particle in which tRNAs serve the function of both substrate and structural foundation for a large molecular machine
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ATP + Asp + tRNAAsn
AMP + diphosphate + aspartyl-tRNAAsn
show the reaction diagram
-
while large amounts of Asp-tRNAAsn are detrimental to Escherichia coli with trypA34 missense mutation, a smaller amount supports protein synthesis and allows the formation of up to 38% of the wild-type level of missense-suppressed tryptophan synthetase
-
-
?
ATP + Asp + tRNAAsn
AMP + diphosphate + aspartyl-tRNAAsn
show the reaction diagram
-
while large amounts pf Asp-tRNAAsn are detrimental to Escherichia coli with trypA34 missense mutation, a smaller amount supports protein synthesis and allows the formation of up to 38% of the wild-type level of missense-suppressed tryptophan synthetase
-
-
?
ATP + L-aspartate + tRNAAsn
AMP + diphosphate + Asp-tRNAAsn
show the reaction diagram
-
only the enzyme AspRS2 aspartylates tRNAAsn
-
-
?
ATP + L-aspartate + tRNAAsn
AMP + diphosphate + L-aspartyl-tRNAAsn
show the reaction diagram
-
-
-
-
?
ATP + L-aspartate + tRNAAsn
AMP + diphosphate + L-aspartyl-tRNAAsn
show the reaction diagram
-, O26328
-
-
-
?
ATP + L-aspartate + tRNAAsn
AMP + diphosphate + L-aspartyl-tRNAAsn
show the reaction diagram
-
the product L-aspartyl-tRNAAsn is transamidated by amidotransferase to form Asn-tRNAAsn. Synthesis of Asn-tRNA via the indirect pathway
-
-
?
ATP + L-aspartate + tRNAAsn
AMP + diphosphate + L-aspartyl-tRNAAsn
show the reaction diagram
-
aspartyl-tRNA synthetase requires a conserved proline, P77, in the anticodon-binding loop for tRNA(Asn) recognition in vivo. Wild-type enzyme shows a slight preference to tRNAAsn over tRNAAsp
-
-
?
ATP + L-aspartate + tRNAAsn
AMP + diphosphate + L-aspartyl-tRNAAsn
show the reaction diagram
-
two residues in the anticodon recognition domain of the aspartyl-tRNA synthetase, H31 and G83, are individually implicated in the recognition of tRNAAsn
-
-
?
ATP + L-aspartate + tRNAAsn
AMP + diphosphate + L-aspartyl-tRNAAsn
show the reaction diagram
-
Thermus thermophilus deprived of asparagine synthetase snthesizes Asn on tRNAAsn via a tRNA-dependent pathway involving a nondiscriminating aspartyl-tRNA synthetase that charges Asp onto tRNAAsn prior to conversion of the Asp to Asn by GatCAB
-
-
?
ATP + L-aspartate + tRNAAsn
AMP + diphosphate + L-aspartyl-tRNAAsn
show the reaction diagram
-
ND-AspRSKtRNAAsn complex and of the transamidosome and mechanism of transamidation, overview. A scaffold tRNAAsn mediates stability and integrity of the complex
-
-
?
ATP + L-aspartate + tRNAAsn
AMP + diphosphate + L-aspartyl-tRNAAsn
show the reaction diagram
Methanothermobacter thermautotrophicus DSM 1053
O26328
-
-
-
?
ATP + L-aspartate + tRNAAsn
AMP + diphosphate + aspartyl-tRNAAsn
show the reaction diagram
-
-
-
-
?
ATP + L-aspartate + tRNAAsn
AMP + diphosphate + aspartyl-tRNAAsn
show the reaction diagram
-
-
-
-
?
ATP + L-aspartate + tRNAAsn
AMP + diphosphate + aspartyl-tRNAAsn
show the reaction diagram
-
dual activity of the ND-AspRS since an asparaginyl-tRNA synthetase is missing in Sulfolobus tokodaii strain 7
-
-
?
ATP + L-aspartate + tRNAAsn
AMP + diphosphate + aspartyl-tRNAAsn
show the reaction diagram
-, O07683
reaction via transamidation mechanism
-
-
?
ATP + L-aspartate + tRNAAsn
AMP + diphosphate + aspartyl-tRNAAsn
show the reaction diagram
-, O07683
in vivo expressed Halobacterium salinarum tRNAAsn, reaction via transamidation mechanism
-
-
?
ATP + L-aspartate + tRNAAsn
AMP + diphosphate + aspartyl-tRNAAsn
show the reaction diagram
-
recombinantly produced tRNA substrate
-
-
?
ATP + L-aspartate + tRNAAsn
AMP + diphosphate + aspartyl-tRNAAsn
show the reaction diagram
-
tRNA substrate from Escherichia coli
-
-
?
ATP + L-aspartate + tRNAAsn
AMP + diphosphate + aspartyl-tRNAAsn
show the reaction diagram
Halobacterium salinarum NRC-1
O07683
reaction via transamidation mechanism, in vivo expressed Halobacterium salinarum tRNAAsn, reaction via transamidation mechanism
-
-
?
ATP + L-aspartate + tRNAAsp
AMP + diphosphate + L-aspartyl-tRNAAsp
show the reaction diagram
-, O26328
-
-
-
?
ATP + L-aspartate + tRNAAsp
AMP + diphosphate + L-aspartyl-tRNAAsp
show the reaction diagram
-
aspartyl-tRNA synthetase requires a conserved proline, P77, in the anticodon-binding loop for tRNA(Asn) recognition in vivo. Wild-type enzyme shows a slight preference to tRNAAsn over tRNAAsp
-
-
?
ATP + L-aspartate + tRNAAsp
AMP + diphosphate + L-aspartyl-tRNAAsp
show the reaction diagram
Methanothermobacter thermautotrophicus DSM 1053
O26328
-
-
-
?
ATP + L-aspartate + tRNAAsp
AMP + diphosphate + Asp-tRNAAsp
show the reaction diagram
-
-
-
-
?
ATP + L-aspartate + tRNAAsp
AMP + diphosphate + Asp-tRNAAsp
show the reaction diagram
-
-
-
-
?
ATP + L-aspartate + tRNAAsp
AMP + diphosphate + Asp-tRNAAsp
show the reaction diagram
-
-
-
-
?
ATP + L-aspartate + tRNAAsp
AMP + diphosphate + Asp-tRNAAsp
show the reaction diagram
-
-
-
-
?
ATP + L-aspartate + tRNAAsp
AMP + diphosphate + aspartyl-tRNAAsp
show the reaction diagram
-
-
-
-
?
ATP + L-aspartate + tRNAAsp
AMP + diphosphate + aspartyl-tRNAAsp
show the reaction diagram
-
-
-
-
?
ATP + L-aspartate + tRNAAsp
AMP + diphosphate + aspartyl-tRNAAsp
show the reaction diagram
-
-
-
-
?
ATP + L-aspartate + tRNAAsp
AMP + diphosphate + aspartyl-tRNAAsp
show the reaction diagram
-, O07683
-
-
-
?
ATP + L-aspartate + tRNAAsp
AMP + diphosphate + aspartyl-tRNAAsp
show the reaction diagram
-, O07683
in vivo expressed Halobacterium salinarum tRNAAsp
-
-
?
ATP + L-aspartate + tRNAAsp
AMP + diphosphate + aspartyl-tRNAAsp
show the reaction diagram
-
recombinantly produced tRNA substrate
-
-
?
ATP + L-aspartate + tRNAAsp
AMP + diphosphate + aspartyl-tRNAAsp
show the reaction diagram
-
tRNA substrate from Escherichia coli
-
-
?
ATP + L-aspartate + tRNAAsp
AMP + diphosphate + aspartyl-tRNAAsp
show the reaction diagram
Halobacterium salinarum NRC-1
O07683
-
-
-
?
ATP + L-aspartate + tRNAAsp
AMP + diphosphate + aspartyl-tRNAAsx
show the reaction diagram
-
preparation of recombinant tRNA substrates from Pseudomonas aeruginosa and Saccharomyces cerevisiae, overview, activity with two variants C36U and C38U of yeast tRNAAsp
-
-
?
ATP + L-aspartate + tRNAAsx
AMP + diphosphate + aspartyl-tRNAAsx
show the reaction diagram
-
-
-
-
?
additional information
?
-
-
in bacteria that lack AsnRS, AspRS is nondiscriminating and generates both Asp-tRNAAsp and the noncanonical, misacylated Asp-tRNAAsn, this misacylated tRNA is subsequently repaired by the glutamine-dependent Asp-tRNAAsn/Glu-tRNAGln amidotransferase, EC 6.3.5.6, increasing tRNAAsp specificity in an ND-AspRS diminishes in vivo toxicity
-
-
-
additional information
?
-
-, O07683
the tRNA-dependent transamidation pathway is the essential route for Asn-tRNAAsn formation in organisms that lack an asparaginyl-tRNA synthetase. This pathway relies on ND-AspRS, an enzyme with relaxed tRNA specificity, to form Asp-tRNAAsn, the misacylated tRNA is then converted to Asn-tRNAAsn by the action of an Asp-tRNAAsn amidotransferase, EC 6.3.5.6
-
-
-
additional information
?
-
-, O07683
the Halobacterium salinarum enzyme is unable to use Escherichia coli tRNA as substrate
-
-
-
additional information
?
-
-
the purified protein has the ability to catalyze the aspartylation of hydroxylamine through an aspartyl-AMP intermediate
-
-
-
additional information
?
-
-
tRNA anticodon binding site structures, overview, Helicobacter pylori AspRS is a nondiscriminating enzyme that aminoacylates both tRNAAsp and tRNAAsn, ND-AspRS is 1.7times more efficient at aminoacylating tRNAAsp over tRNAAsn
-
-
-
additional information
?
-
-
two residues in the anticodon recognition domain of the enzyme are individually implicated in the recognition of tRNAAsn, nondiscriminating AspRSs possess a histidine at position 31 and usually a glycine at position 83, whereas discriminating AspRSs, EC 6.1.1.12, possess a leucine at position 31 and a residue other than a glycine at position 83
-
-
-
additional information
?
-
-
mutations in the DARS2 gene lead to Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation as a disorder with an autosomal recessive mode of inheritance, the phenotype includes cerebellar, pyramidal and dorsal column dysfunctions, overview
-
-
-
additional information
?
-
-
the enzyme is downregulated at the post-transcriptional level in a complex retro-inhibition mechanism, the cell equilibrates cellular concentrations of tRNAAsp, AspRS, and its encoding mRNA to hinder AspRS accumulation and avoid misacylation of heterologous tRNAs, proteomic analysis, overview
-
-
-
additional information
?
-
C4LZN0
AspRS must perform two sequential reactions. The first reaction is the formation of aspartyladenylate from the free amino acid and ATP, releasing diphosphate. The second reaction is the displacement of the adenylate moiety by the 3'-OH of the terminal adenosine of the tRNAAsp acceptor arm, yielding the covalently linked Asp-tRNAAsp. The active site must therefore bind the two initial reactants, Asp and ATP, and also provide access for the properly positioned acceptor stem of a bound tRNA molecule
-
-
-
additional information
?
-
Halobacterium salinarum NRC-1
O07683
the tRNA-dependent transamidation pathway is the essential route for Asn-tRNAAsn formation in organisms that lack an asparaginyl-tRNA synthetase. This pathway relies on ND-AspRS, an enzyme with relaxed tRNA specificity, to form Asp-tRNAAsn, the misacylated tRNA is then converted to Asn-tRNAAsn by the action of an Asp-tRNAAsn amidotransferase, EC 6.3.5.6, the Halobacterium salinarum enzyme is unable to use Escherichia coli tRNA as substrate
-
-
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
ATP + Asp + tRNAAsn
AMP + diphosphate + aspartyl-tRNAAsn
show the reaction diagram
-
while large amounts of Asp-tRNAAsn are detrimental to Escherichia coli with trypA34 missense mutation, a smaller amount supports protein synthesis and allows the formation of up to 38% of the wild-type level of missense-suppressed tryptophan synthetase
-
-
?
ATP + Asp + tRNAAsn
AMP + diphosphate + aspartyl-tRNAAsn
show the reaction diagram
-
while large amounts pf Asp-tRNAAsn are detrimental to Escherichia coli with trypA34 missense mutation, a smaller amount supports protein synthesis and allows the formation of up to 38% of the wild-type level of missense-suppressed tryptophan synthetase
-
-
?
ATP + L-aspartate + tRNAAsn
AMP + diphosphate + L-aspartyl-tRNAAsn
show the reaction diagram
-
-
-
-
?
ATP + L-aspartate + tRNAAsn
AMP + diphosphate + L-aspartyl-tRNAAsn
show the reaction diagram
-, O26328
-
-
-
?
ATP + L-aspartate + tRNAAsn
AMP + diphosphate + L-aspartyl-tRNAAsn
show the reaction diagram
-
the product L-aspartyl-tRNAAsn is transamidated by amidotransferase to form Asn-tRNAAsn. Synthesis of Asn-tRNA via the indirect pathway
-
-
?
ATP + L-aspartate + tRNAAsn
AMP + diphosphate + L-aspartyl-tRNAAsn
show the reaction diagram
-
Thermus thermophilus deprived of asparagine synthetase snthesizes Asn on tRNAAsn via a tRNA-dependent pathway involving a nondiscriminating aspartyl-tRNA synthetase that charges Asp onto tRNAAsn prior to conversion of the Asp to Asn by GatCAB
-
-
?
ATP + L-aspartate + tRNAAsn
AMP + diphosphate + aspartyl-tRNAAsn
show the reaction diagram
-
-
-
-
?
ATP + L-aspartate + tRNAAsn
AMP + diphosphate + aspartyl-tRNAAsn
show the reaction diagram
-
-
-
-
?
ATP + L-aspartate + tRNAAsn
AMP + diphosphate + aspartyl-tRNAAsn
show the reaction diagram
-
dual activity of the ND-AspRS since an asparaginyl-tRNA synthetase is missing in Sulfolobus tokodaii strain 7
-
-
?
ATP + L-aspartate + tRNAAsn
AMP + diphosphate + aspartyl-tRNAAsn
show the reaction diagram
-, O07683
reaction via transamidation mechanism
-
-
?
ATP + L-aspartate + tRNAAsn
AMP + diphosphate + L-aspartyl-tRNAAsn
show the reaction diagram
Methanothermobacter thermautotrophicus DSM 1053
O26328
-
-
-
?
ATP + L-aspartate + tRNAAsn
AMP + diphosphate + aspartyl-tRNAAsn
show the reaction diagram
Halobacterium salinarum NRC-1
O07683
reaction via transamidation mechanism
-
-
?
ATP + L-aspartate + tRNAAsp
AMP + diphosphate + L-aspartyl-tRNAAsp
show the reaction diagram
-, O26328
-
-
-
?
ATP + L-aspartate + tRNAAsp
AMP + diphosphate + Asp-tRNAAsp
show the reaction diagram
-
-
-
-
?
ATP + L-aspartate + tRNAAsp
AMP + diphosphate + Asp-tRNAAsp
show the reaction diagram
-
-
-
-
?
ATP + L-aspartate + tRNAAsp
AMP + diphosphate + Asp-tRNAAsp
show the reaction diagram
-
-
-
-
?
ATP + L-aspartate + tRNAAsp
AMP + diphosphate + aspartyl-tRNAAsp
show the reaction diagram
-
-
-
-
?
ATP + L-aspartate + tRNAAsp
AMP + diphosphate + aspartyl-tRNAAsp
show the reaction diagram
-
-
-
-
?
ATP + L-aspartate + tRNAAsp
AMP + diphosphate + aspartyl-tRNAAsp
show the reaction diagram
-
-
-
-
?
ATP + L-aspartate + tRNAAsp
AMP + diphosphate + aspartyl-tRNAAsp
show the reaction diagram
-, O07683
-
-
-
?
ATP + L-aspartate + tRNAAsp
AMP + diphosphate + L-aspartyl-tRNAAsp
show the reaction diagram
Methanothermobacter thermautotrophicus DSM 1053
O26328
-
-
-
?
ATP + L-aspartate + tRNAAsp
AMP + diphosphate + Asp-tRNAAsp
show the reaction diagram
-
-
-
-
?
ATP + L-aspartate + tRNAAsp
AMP + diphosphate + aspartyl-tRNAAsp
show the reaction diagram
Halobacterium salinarum NRC-1
O07683
-
-
-
?
ATP + L-aspartate + tRNAAsx
AMP + diphosphate + aspartyl-tRNAAsx
show the reaction diagram
-
-
-
-
?
additional information
?
-
-
in bacteria that lack AsnRS, AspRS is nondiscriminating and generates both Asp-tRNAAsp and the noncanonical, misacylated Asp-tRNAAsn, this misacylated tRNA is subsequently repaired by the glutamine-dependent Asp-tRNAAsn/Glu-tRNAGln amidotransferase, EC 6.3.5.6, increasing tRNAAsp specificity in an ND-AspRS diminishes in vivo toxicity
-
-
-
additional information
?
-
-, O07683
the tRNA-dependent transamidation pathway is the essential route for Asn-tRNAAsn formation in organisms that lack an asparaginyl-tRNA synthetase. This pathway relies on ND-AspRS, an enzyme with relaxed tRNA specificity, to form Asp-tRNAAsn, the misacylated tRNA is then converted to Asn-tRNAAsn by the action of an Asp-tRNAAsn amidotransferase, EC 6.3.5.6
-
-
-
additional information
?
-
-
mutations in the DARS2 gene lead to Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation as a disorder with an autosomal recessive mode of inheritance, the phenotype includes cerebellar, pyramidal and dorsal column dysfunctions, overview
-
-
-
additional information
?
-
-
the enzyme is downregulated at the post-transcriptional level in a complex retro-inhibition mechanism, the cell equilibrates cellular concentrations of tRNAAsp, AspRS, and its encoding mRNA to hinder AspRS accumulation and avoid misacylation of heterologous tRNAs, proteomic analysis, overview
-
-
-
additional information
?
-
C4LZN0
AspRS must perform two sequential reactions. The first reaction is the formation of aspartyladenylate from the free amino acid and ATP, releasing diphosphate. The second reaction is the displacement of the adenylate moiety by the 3'-OH of the terminal adenosine of the tRNAAsp acceptor arm, yielding the covalently linked Asp-tRNAAsp. The active site must therefore bind the two initial reactants, Asp and ATP, and also provide access for the properly positioned acceptor stem of a bound tRNA molecule
-
-
-
additional information
?
-
Halobacterium salinarum NRC-1
O07683
the tRNA-dependent transamidation pathway is the essential route for Asn-tRNAAsn formation in organisms that lack an asparaginyl-tRNA synthetase. This pathway relies on ND-AspRS, an enzyme with relaxed tRNA specificity, to form Asp-tRNAAsn, the misacylated tRNA is then converted to Asn-tRNAAsn by the action of an Asp-tRNAAsn amidotransferase, EC 6.3.5.6
-
-
-
COFACTOR
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
Mg2+
-
3 mol per mol of enzyme
Mg2+
-
required
NaCl
-, O07683
required at 0.1-3 M, salt dependence profile, overview
INHIBITORS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
heptapeptide-nucleotide microcin C
-
McC, an antimicrobial nucleotide peptide that targets and strongly inhibits aspartyl-tRNA synthetase. The inhibitor cannot be processed by the nonspecific oligopeptidases PepA, PepB, or PepN, the rate-limiting step of McC processing in vitro is deformylation of the first methionine residue of McC, structure, massspectrometric analysis of McC produced by wild-type Escherichia coli cells, overview
L-aspartol adenylate
-
i.e. Asp-ol-AMP, a stable analogue of the natural reaction intermediate L-aspartyl adenylate, biphasic, competitive inhibition, differential inhibition of tRNAAsp and tRNAAsn aspartylation by the enzyme, overview
ACTIVATING COMPOUND
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
KM VALUE [mM]
KM VALUE [mM] Maximum
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.005
-
aspartate
-
AspRS2
0.03
-
aspartate
-
AspRS1
0.033
-
ATP
-
AspRS2
0.00006
-
tRNAAsn
-
AspRS2
0.002
-
tRNAAsn
-
37C, pH 7.0, wild-type enzym
0.0035
-
tRNAAsn
-
37C, pH 7.0, mutant enzyme P77K/H28Q
0.0038
-
tRNAAsn
-
37C, pH 7.0, mutant enzyme P77K
0.0045
-
tRNAAsn
-
37C, pH 7.0, mutant enzyme H28Q
0.83
-
tRNAAsn
-
pH 7.5, recombinant wild-type enzyme
1.87
-
tRNAAsn
-
pH 7.5, recombinant mutant L81N enzyme
2.23
-
tRNAAsn
-
pH 7.5, recombinant mutant L86M enzyme
0.00003
-
tRNAAsp
-
AspRS1
0.00007
-
tRNAAsp
-
AspRS2
0.0008
-
tRNAAsp
-
37C, pH 7.0, mutant enzyme P77K; 37C, pH 7.0, wild-type enzyme
0.0013
-
tRNAAsp
-
37C, pH 7.0, mutant enzyme P77K/H28Q
0.0018
-
tRNAAsp
-
37C, pH 7.0, mutant enzyme H28Q
0.77
-
tRNAAsp
-
pH 7.5, recombinant wild-type enzyme
0.85
-
tRNAAsp
-
pH 7.5, recombinant mutant L81N enzyme
0.87
-
tRNAAsp
-
pH 7.5, recombinant mutant L86M enzyme
0.28
-
ATP
-
AspRS1
additional information
-
additional information
-
-
-
additional information
-
additional information
-
the L81N/L86M mutant does not follow Michaelis-Menten kinetics
-
additional information
-
additional information
-
pre-steady-state and steady-state aminoacylation kinetics of the ND-AspRSK-tRNAAsn complex and of the transamidosome, overview
-
TURNOVER NUMBER [1/s]
TURNOVER NUMBER MAXIMUM[1/s]
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.014
-
tRNAAsn
-
pH 7.5, recombinant wild-type enzyme
0.015
-
tRNAAsn
-
pH 7.5, recombinant mutant L86M enzyme
0.026
-
tRNAAsn
-
pH 7.5, recombinant mutant L81N enzyme
0.092
-
tRNAAsn
-
AspRS2
0.22
-
tRNAAsn
-
37C, pH 7.0, mutant enzyme H28Q; 37C, pH 7.0, mutant enzyme P77K/H28Q
0.51
-
tRNAAsn
-
37C, pH 7.0, wild-type enzym
0.6
-
tRNAAsn
-
37C, pH 7.0, mutant enzyme P77K
0.021
-
tRNAAsp
-
pH 7.5, recombinant mutant L86M enzyme
0.022
-
tRNAAsp
-
pH 7.5, recombinant wild-type enzyme
0.028
-
tRNAAsp
-
pH 7.5, recombinant mutant L81N enzyme
0.14
-
tRNAAsp
-
37C, pH 7.0, mutant enzyme P77K/H28Q
0.15
-
tRNAAsp
-
37C, pH 7.0, wild-type enzyme
0.17
-
tRNAAsp
-
37C, pH 7.0, mutant enzyme H28Q
0.24
-
tRNAAsp
-
AspRS2
0.28
-
tRNAAsp
-
37C, pH 7.0, mutant enzyme P77K
7.2
-
tRNAAsp
-
AspRS1
additional information
-
additional information
-
the wild-type enzyme has a kcat for tRNAAsp that is 60% higher than that of tRNAAsn
-
Ki VALUE [mM]
Ki VALUE [mM] Maximum
INHIBITOR
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.041
-
L-aspartol adenylate
-
tRNAAsp aspartylation
0.215
-
L-aspartol adenylate
-
tRNAAsn aspartylation
additional information
-
additional information
-
inhibition kinetics
-
SPECIFIC ACTIVITY [µmol/min/mg]
SPECIFIC ACTIVITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
additional information
-
-
activities of wild-type and mutant enzymes with different tRNA substrates
pH OPTIMUM
pH MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
7.2
-
-, O07683
aminoacylation assay at
7.2
-
-
activity measurement of the transamidosome
7.2
-
-
assay at
7.5
-
-
assay at
7.5
-
-
assay at
7.5
-
-
assay at
TEMPERATURE OPTIMUM
TEMPERATURE OPTIMUM MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
25
-
-
assay at
37
-
-
assay at
37
-
-, O07683
assay at
37
-
-
activity measurement of the transamidosome
TEMPERATURE RANGE
TEMPERATURE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
10
85
-
transamidosome complex: ND-AspRS is thermostable up to 70C, but its thermostability increases when complexed to tRNAAsn. tRNAAsn is very stable, as its melting temperature is 85C. the GatCAB is poorly protected against heat inactivation, as its denaturation starts at 40C, but when complexed in the transamidosome, the GatCAB becomes fully thermostable at 85C
SOURCE TISSUE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
-
the partners involved in tRNA-dependent Asn formation assemble into a ternary complex called the transamidosome, consisting of the ND-AspRS, GatCAD amidotransferase, and tRNAAsn
-
Manually annotated by BRENDA team
MOLECULAR WEIGHT
MOLECULAR WEIGHT MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
300000
-
-
ternary complex, transamidosome, ND-AspRS-GatCAB-tRNAAsn, theoretical
380000
-
-
ternary complex, transamidosome, ND-AspRS-GatCAB-tRNAAsn, determined by gel filtration
400000
-
-
ternary complex, transamidosome, ND-AspRS-GatCAB-tRNAAsn, determined by static light scattering, SLS
SUBUNITS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
?
-
x * 49074, sequence calculation
trimer
-
ternary complex, transamidosome, ND-AspRS-GatCAB-tRNAAsn
homodimer
C4LZN0
structure modelling, overview
additional information
-
structural comparison of the nondiscriminating AspRS with structures of discriminating AspRSs, EC 6.1.1.12, highly conserved catalytic domain, but different tRNA binding site in the N-terminal domain, overview
additional information
C4LZN0
AspRS is a class IIb aminoacyl-tRNA synthetase showing conserved structural features, overview
additional information
-
comparison of three-dimensional transamidosome complex structures in crystals and in solution, overview
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
additional information
-
post-translational modifications in the N-terminal extension of AspRS neutralizing the lysine-rich motif contained in this domain
Crystallization/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
purified AspRS, sitting drop vapour diffusion method, 0.0002 ml of 9 mg/ml protein is mixed with 0.0002 ml of reservoir solution containing 0.2 M lithium sulfate, 0.1 M Bis-Tris, pH 5.5, and 23% w/v PEG 3350, X-ray diffraction structure determination and analysis at 2.8 A resolution
C4LZN0
purified recombinant enzyme, 5-8 mg/ml in 20 mM TrisHCl buffer, pH 7.0, hanging-drop vapour-diffusion method, 26C, from 0.002 ml protein solution is mixed with 0.002 ml of reservoir solution containing 100 mM sodium HEPES buffer, pH 7.5, containing 100 mM NaCl and 1.6 M (NH4)2SO4, equilibration against 0.7 ml reservoir solution, X-ray diffraction structure determination and analysis at 2.3 A resolution, molecular replacement
-
purified recombinant enzyme, X-ray diffraction structure determination and analysis at 2.3 A resolution
-
hanging drop technique, 24C, 5 days in Crystal Screen I or II, solutions containing either polyethylene glycol or ethylene glycol
-
a complete dataset of the transamidosome is collected to 3 A resolution
-
purified 520 kDa transamidosome complex formed by two dimeric nondiscriminating-AspRSs, two trimeric GatCABs, and four tRNAsAsn molecules, X-ray diffraction structure determination and analysis at 3.0 A resolution, molecular replacement
-
GENERAL STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
ND-AspRS is stable in low and high salt
-, O07683
STORAGE STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
-20C, purified recombinant enzyme, in 50% glycerol, and 33 mM phosphate, pH 7.4, 3 mM Tris-HCl, 1.5 mM 2-mercaptoethanol, and 0.5 mM phenylmethanesulfonyl fluoride, stable for months
-
Purification/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
recombinant His-tagged AspRS, cleavage of the N-terminal His-tag
C4LZN0
recombinant His-tagged wild-type and mutant ND-AspRS in Escherichia coli strain trpA34 by nickel affinity chromatography
-, O07683
recombinant His-tagged AsnRS from Escherichia coli strain DH5alpha by nickel affinity chromatography
-
recombinant His-tagged enzyme from strain ADD1976 by nickel affinity chromatography
-
recombinant enzyme from Escherichia coli strain BL21(DE3)
-
recombinant enzyme from Escherichia coli strain BL21(DE3) by anion exchange and hydroxyapatite chromatography
-
recombinant enzyme
-
recombinant enzyme
-
the transamidosome complex is isolated by preparative gel filtration on a Superdex G200 column
-
Cloned/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
Escherichia coli trypA34 missense mutant transformed with heterologous ND-aspS gene
-
expression in Escherichia coli
-
recombinant expression of the N-terminally His-tagged AspRS
C4LZN0
Escherichia coli trypA34 missense mutant transformed with heterologous ND-aspS gene
-
gene aspS, phylogenetic tree, expression of His-tagged wild-type and mutant ND-AspRS in Escherichia coli strain trpA34, carrying a D60N mutation in trpA leading to tryptophan auxotrophy, co-expression of tRNAAsn leading to restoration of tryptophan prototrophy by missense suppression of the trpA34 mutant with heterologously in vivo formed Asp-tRNAAsn
-, O07683
overexpression of His-tagged AsnRS in Escherichia coli strain DH5alpha, the enzyme is toxic when heterologously overexpressed in Escherichia coli, because of sequestration of tRNAAsn as Asp-tRNAAsn, this toxicity is rescued upon coexpression of the Helicobacter pylori Asp/Glu-Adt, EC 6.3.5.6
-
gene DARS2, DNA and amino acid analysis of wild-type and mutant enzyme
-
expression in Escherichia coli
O26328
expression of C-terminally His-tagged enzyme in strain ADD1976
-
gene aspS, DNA and amino acid sequence determination and analysis, expression of His-tagged wild-type and mutant enzymes
-
overproduced from the cloned gene in Pseudomonas aeruginosa
-
expression in YBC-603 cells
-
overexpression in Escherichia coli strain BL21(DE3)
-
expression in JM101Tr cells
-
for expression in Escherichia coli cells
-
overexpression in Escherichia coli
-
ENGINEERING
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
H28Q
-
wild-type enzyme shows a slight preference to tRNAAsn over tRNAAsp. Mutation H28Q leads to a reverse tRNA preference
H77K/H28Q
-
wild-type enzyme shows a slight preference to tRNAAsn over tRNAAsp. Mutation P77K/H28Q leads to a reverse tRNA preference
P77K
-
wild-type enzyme shows a slight preference to tRNAAsn over tRNAAsp. Mutation P77K leads to a reverse tRNA preference and a 3fold increase in specificity for tRNAASp over tRNAAsn
H26A
-, O07683
site-directed mutagenesis, mutation the amino acid located in the AspRS anticodon binding domain limits the specificity of this nondiscriminating enzyme towards tRNAAsn, altered tRAN substrate specificity compared to the wild-type enzyme, overview
H26A/P84A
-, O07683
site-directed mutagenesis, mutation the amino acid located in the AspRS anticodon binding domain limits the specificity of this nondiscriminating enzyme towards tRNAAsn, altered tRAN substrate specificity compared to the wild-type enzyme, overview
H26A/P84K
-, O07683
site-directed mutagenesis, mutation the amino acid located in the AspRS anticodon binding domain limits the specificity of this nondiscriminating enzyme towards tRNAAsn, altered tRAN substrate specificity compared to the wild-type enzyme, overview
H26Q
-, O07683
site-directed mutagenesis, mutation the amino acid located in the AspRS anticodon binding domain limits the specificity of this nondiscriminating enzyme towards tRNAAsn, altered tRAN substrate specificity compared to the wild-type enzyme, overview
H26Q/P84A
-, O07683
site-directed mutagenesis, mutation the amino acid located in the AspRS anticodon binding domain limits the specificity of this nondiscriminating enzyme towards tRNAAsn, altered tRAN substrate specificity compared to the wild-type enzyme, overview
H26Q/P84K
-, O07683
site-directed mutagenesis, mutation the amino acid located in the AspRS anticodon binding domain limits the specificity of this nondiscriminating enzyme towards tRNAAsn, altered tRAN substrate specificity compared to the wild-type enzyme, overview
P84A
-, O07683
site-directed mutagenesis, mutation the amino acid located in the AspRS anticodon binding domain limits the specificity of this nondiscriminating enzyme towards tRNAAsn, altered tRAN substrate specificity compared to the wild-type enzyme, overview
P84K
-, O07683
site-directed mutagenesis, mutation the amino acid located in the AspRS anticodon binding domain limits the specificity of this nondiscriminating enzyme towards tRNAAsn, altered tRAN substrate specificity compared to the wild-type enzyme, overview
H26A
Halobacterium salinarum NRC-1
-
site-directed mutagenesis, mutation the amino acid located in the AspRS anticodon binding domain limits the specificity of this nondiscriminating enzyme towards tRNAAsn, altered tRAN substrate specificity compared to the wild-type enzyme, overview
-
H26Q
Halobacterium salinarum NRC-1
-
site-directed mutagenesis, mutation the amino acid located in the AspRS anticodon binding domain limits the specificity of this nondiscriminating enzyme towards tRNAAsn, altered tRAN substrate specificity compared to the wild-type enzyme, overview
-
H26Q/P84A
Halobacterium salinarum NRC-1
-
site-directed mutagenesis, mutation the amino acid located in the AspRS anticodon binding domain limits the specificity of this nondiscriminating enzyme towards tRNAAsn, altered tRAN substrate specificity compared to the wild-type enzyme, overview
-
P84A
Halobacterium salinarum NRC-1
-
site-directed mutagenesis, mutation the amino acid located in the AspRS anticodon binding domain limits the specificity of this nondiscriminating enzyme towards tRNAAsn, altered tRAN substrate specificity compared to the wild-type enzyme, overview
-
P84K
Halobacterium salinarum NRC-1
-
site-directed mutagenesis, mutation the amino acid located in the AspRS anticodon binding domain limits the specificity of this nondiscriminating enzyme towards tRNAAsn, altered tRAN substrate specificity compared to the wild-type enzyme, overview
-
L81N
-
site-directed mutagenesis, the mutation in the anticodon binding domain doubles the kcat for tRNAAsn as compared to the wild-type enzyme
L81N/L86M
-
site-directed mutagenesis, the mutation in the anticodon binding domain alters the tRNA specificity as compared to the wild-type enzyme, the L81N/L86M mutant does not follow Michaelis-Menten kinetics
G83K
-
aspartylation reaction of the mutant enzyme is 55% as fast as the wild-type enzyme; site-directed mutagenesis, the mutation increases the specificity of tRNAAsp charging over that of tRNAAsn by 4.2fold
R485K
-
a catalytic site mutant
L86M
-
site-directed mutagenesis, the mutation in the anticodon binding domain alters the tRNA specificity as compared to the wild-type enzyme
additional information
-
mutations in the anticodon binding domain of Helicobacter pylori ND-AspRS, e.g. at L81, L86, N82, and M87, reduce this enzymes ability to misacylate tRNAAsn and enhance tRNAAsp specificity, in a manner that correlates with the toxicity of the enzyme in Escherichia coli, overview
additional information
-
naturylla occuring gene DARS2 mutant phenotype, overview
H31L
-
aspartylation reaction of the mutant enzyme is 84% as fast as the wild-type enzyme; aspartylation reaction of the mutant enzyme is 92% as fast as the wild-type enzyme; site-directed mutagenesis, the mutation increases the specificity of tRNAAsp charging over that of tRNAAsn by 3.5fold
additional information
-
AspRS overexpression leads deprivation of the gene's 5'-untranslated region, that is essential for directing mRNA recognition by the protein and initiating the retro-inhibition process, but does not lead to increased tRNAAsn and/or tRNAGlu misaspartylation or the logical consecutive post-translational stress