Information on EC 6.3.5.7 - glutaminyl-tRNA synthase (glutamine-hydrolysing)

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

EC NUMBER
COMMENTARY hide
6.3.5.7
-
RECOMMENDED NAME
GeneOntology No.
glutaminyl-tRNA synthase (glutamine-hydrolysing)
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + L-glutamyl-tRNAGln + L-glutamine = ADP + phosphate + L-glutaminyl-tRNAGln + L-glutamate
show the reaction diagram
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Aminoacyl-tRNA biosynthesis
-
-
glutamate and glutamine metabolism
-
-
glutaminyl-tRNAgln biosynthesis via transamidation
-
-
Metabolic pathways
-
-
SYSTEMATIC NAME
IUBMB Comments
glutamyl-tRNAGln:L-glutamine amido-ligase (ADP-forming)
In systems lacking discernible glutamine---tRNA ligase (EC 6.1.1.18), glutaminyl-tRNAGln is formed by a two-enzyme system. In the first step, a nondiscriminating ligase (EC 6.1.1.24, glutamate---tRNAGln ligase) mischarges tRNAGln with glutamate, forming glutamyl-tRNAGln. The glutamyl-tRNAGln is not used in protein synthesis until the present enzyme converts it into glutaminyl-tRNAGln (glutamyl-tRNAGlu is not a substrate for this reaction). Ammonia or asparagine can substitute for the preferred substrate glutamine.
CAS REGISTRY NUMBER
COMMENTARY hide
52232-48-1
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
mutant C-2A
-
-
Manually annotated by BRENDA team
Methanothermobacter thermautotrophicum
-
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ATP + Asp-tRNAAsn + L-glutamine
ADP + phosphate + Asn-tRNAAsn + L-glutamate
show the reaction diagram
ATP + Glu-tRNAGln + Asn
ADP + phosphate + Gln-tRNAGln + Asp
show the reaction diagram
ATP + Glu-tRNAGln + L-asparagine
ADP + phosphate + Gln-tRNAGln + L-glutamate
show the reaction diagram
-
-
-
-
?
ATP + Glu-tRNAGln + L-glutamine
ADP + phosphate + Gln-tRNAGln + L-glutamate
show the reaction diagram
ATP + Glu-tRNAGln + L-glutamine + H2O
ADP + phosphate + Gln-tRNAGln + L-glutamate
show the reaction diagram
ATP + Glu-tRNAGln + NH4Cl
ADP + phosphate + Gln-tRNAGln + ?
show the reaction diagram
ATP + L-glutamate + tRNAGln1(UUG)
AMP + diphosphate + L-glutamyl-tRNAGln1(UUG)
show the reaction diagram
Methanothermobacter thermautotrophicum
-
the enzyme is active toward the two tRNAGln isoacceptors, but with a significant catalytic preference for tRNAGln2(CUG). The less active tRNAGln1(UUG) responds to the less common CAA codon for Gln
-
-
?
ATP + L-glutamate + tRNAGln2(CUG)
AMP + diphosphate + L-glutamyl-tRNAGln2(CUG)
show the reaction diagram
Methanothermobacter thermautotrophicum
-
the enzyme is active toward the two tRNAGln isoacceptors, but with a significant catalytic preference for tRNAGln2(CUG). The less active tRNAGln1(UUG) responds to the less common CAA codon for Gln. The wild-type enzyme shows a 24fold catalytic preference for tRNAGln2 over tRNAGlu
-
-
?
ATP + L-glutamate + tRNAGlu
AMP + diphosphate + L-glutamyl-tRNAGlu
show the reaction diagram
Methanothermobacter thermautotrophicum
-
-
-
-
?
ATP-gammaS + Glu-tRNAGln + L-glutamine
? + phosphate + Gln-tRNAGln + L-glutamate
show the reaction diagram
-
-
-
?
additional information
?
-
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
ATP + Asp-tRNAAsn + L-glutamine
ADP + phosphate + Asn-tRNAAsn + L-glutamate
show the reaction diagram
-
-
-
-
?
ATP + Glu-tRNAGln + L-glutamine
ADP + phosphate + Gln-tRNAGln + L-glutamate
show the reaction diagram
ATP + Glu-tRNAGln + L-glutamine + H2O
ADP + phosphate + Gln-tRNAGln + L-glutamate
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(1R,2R)-1-(4-methylsulfonylphenyl)-2-(S-dioxo-L-methioneamido)-1,3-propanediol
-
-
(1R,2R)-1-(4-nitrophenyl)-2-(S-dioxo-L-methioneamido)-1,3-propanediol
-
-
(1R,2R)-1-phenyl-2-(S-dioxo-L-methioneamido)-1,3-propanediol
-
-
(1R,2S)-1-(4-nitrophenyl)-2-(S-dioxo-L-methioneamido)-1,3-propanediol
-
-
(1S,2R)-1-(4-nitrophenyl)-2-(S-dioxo-L-methioneamido)-1,3-propanediol
-
-
(1S,2S)-1-(4-nitrophenyl)-2-(S-dioxo-L-methioneamido)-1,3-propanediol
-
-
2'-O-(trinitrophenyl)adenosine 5'-triphosphate
-
IC50: 2.4 mM
3'-(L-alpha-aspartylamino)-3'-deoxy-N,N-dimethyladenosine
-
puromycin analogue
3'-deoxy-3'-(L-alpha-glutamylamino)-N,N-dimethyladenosine
-
puromycin analogue
3'-deoxy-3'-(L-glutaminylamino)-N,N-dimethyladenosine
-
puromycin analogue
3'-O-(trinitrophenyl)adenosine 5'-triphosphate
-
IC50: 2.4 mM
3'-[[(2S)-2-amino-4-(methylsulfinyl)butanoyl]amino]-3'-deoxy-N,N-dimethyladenosine
-
puromycin analogue
3'-[[(2S)-2-amino-4-(methylsulfonyl)butanoyl]amino]-3'-deoxy-N,N-dimethyladenosine
-
puromycin analogue
3'-[[(2S)-4-carboxy-2-hydroxybutanoyl]amino]-3'-deoxy-N,N-dimethyladenosine
-
puromycin analogue
3'-[[2-amino-4-(methylphosphinato)butanoyl]amino]-3'-deoxy-N,N-dimethyladenosine
-
puromycin analogue
6-diazo-5-oxonorleucine
-
blocking of glutamine-dependent reaction, no inhibition of ammonia-dependent reaction
adenosine 5'-[beta,gamma-methylene]triphosphate
-
IC50: 2.3 mM
ADP
-
IC50: 0.026 mM
ATP-gammaS
-
IC50: 0.19 mM
Chloramphenicol
-
-
gamma-Glu boronic acid
-
IC50: 0.0016 mM
L-methionine-S-sulfoximine
-
at 1 mM, 3 mM or 5 mM, 20% inhibition
puromycin
-
aminonucleoside antibiotic produced by Streptomyces alboniger, very weak inhibitor of AdT
additional information
-
no inhibition by 6-diazo-5-oxonorleucine
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0224 - 0.2
Asn
0.00095
Asp-tRNAAsn
-
37C, pH 7.2, amidotransferase activity
0.00118
Asp-tRNAGln
-
37C, pH 7.2, amidotransferase activity
0.117 - 0.2068
ATP
0.01 - 0.0509
Gln
0.00042 - 0.0024
Glu-tRNAGln
0.0002
L-glutamyl-tRNAGlu
-
-
0.00539
tRNAGln1(UUG)
Methanothermobacter thermautotrophicum
-
pH 7.2, 37C
-
0.00133
tRNAGln2(CUG)
Methanothermobacter thermautotrophicum
-
pH 7.2, 37C
-
0.00361
tRNAGlu
Methanothermobacter thermautotrophicum
-
pH 7.2, 37C
additional information
additional information
Methanothermobacter thermautotrophicum
-
aminoacylation kinetics of enzyme variants
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.027
Asn
Helicobacter pylori
-
37C, pH 7.2, amidotransferase activity
1.3
Asp-tRNAAsn
Helicobacter pylori
-
37C, pH 7.2, amidotransferase activity
3.61
Asp-tRNAGln
Helicobacter pylori
-
37C, pH 7.2, amidotransferase activity
0.59 - 6.1
ATP
0.052 - 11.8
Gln
0.21 - 1.2
Glu-tRNAGln
0.006
tRNAGln1(UUG)
Methanothermobacter thermautotrophicum
-
pH 7.2, 37C
-
0.41
tRNAGln2(CUG)
Methanothermobacter thermautotrophicum
-
pH 7.2, 37C
-
0.04
tRNAGlu
Methanothermobacter thermautotrophicum
-
pH 7.2, 37C
additional information
additional information
Methanothermobacter thermautotrophicum
-
aminoacylation kinetics of enzyme variants
-
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
400 - 600
Glu-tRNAGln
1.1
tRNAGln1(UUG)
Methanothermobacter thermautotrophicum
-
pH 7.2, 37C
194913
308
tRNAGln2(CUG)
Methanothermobacter thermautotrophicum
-
pH 7.2, 37C
194912
11
tRNAGlu
Methanothermobacter thermautotrophicum
-
pH 7.2, 37C
1988
additional information
additional information
Methanothermobacter thermautotrophicum
-
aminoacylation kinetics of enzyme variants
2
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.12
(1R,2R)-1-(4-methylsulfonylphenyl)-2-(S-dioxo-L-methioneamido)-1,3-propanediol
-
pH 7, temperature not specified in the publication
0.027
(1R,2R)-1-(4-nitrophenyl)-2-(S-dioxo-L-methioneamido)-1,3-propanediol
-
pH 7, temperature not specified in the publication
0.4
(1R,2R)-1-phenyl-2-(S-dioxo-L-methioneamido)-1,3-propanediol
-
pH 7, temperature not specified in the publication
0.37
(1R,2S)-1-(4-nitrophenyl)-2-(S-dioxo-L-methioneamido)-1,3-propanediol
-
pH 7, temperature not specified in the publication
2.8
(1S,2R)-1-(4-nitrophenyl)-2-(S-dioxo-L-methioneamido)-1,3-propanediol
-
pH 7, temperature not specified in the publication
0.16
(1S,2S)-1-(4-nitrophenyl)-2-(S-dioxo-L-methioneamido)-1,3-propanediol
-
pH 7, temperature not specified in the publication
0.134
3'-(L-alpha-aspartylamino)-3'-deoxy-N,N-dimethyladenosine
-
-
0.105
3'-deoxy-3'-(L-alpha-glutamylamino)-N,N-dimethyladenosine
-
-
0.045
3'-deoxy-3'-(L-glutaminylamino)-N,N-dimethyladenosine
-
-
0.011
3'-[[(2S)-2-amino-4-(methylsulfinyl)butanoyl]amino]-3'-deoxy-N,N-dimethyladenosine
-
-
0.004
3'-[[(2S)-2-amino-4-(methylsulfonyl)butanoyl]amino]-3'-deoxy-N,N-dimethyladenosine
-
-
0.13
3'-[[(2S)-4-carboxy-2-hydroxybutanoyl]amino]-3'-deoxy-N,N-dimethyladenosine
-
-
0.033
3'-[[2-amino-4-(methylphosphinato)butanoyl]amino]-3'-deoxy-N,N-dimethyladenosine
-
-
1.85
Chloramphenicol
-
pH 7, temperature not specified in the publication
4.1
puromycin
-
very weak inhibitor of AdT
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2.4
2'-O-(trinitrophenyl)adenosine 5'-triphosphate
2.3
adenosine 5'-[beta,gamma-methylene]triphosphate
Streptococcus pyogenes
-
IC50: 2.3 mM
0.026
ADP
Streptococcus pyogenes
-
IC50: 0.026 mM
0.19
ATP-gammaS
Streptococcus pyogenes
-
IC50: 0.19 mM
0.0016
gamma-Glu boronic acid
Streptococcus pyogenes
-
IC50: 0.0016 mM
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.2
Methanothermobacter thermautotrophicum
-
assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
Methanothermobacter thermautotrophicum
-
assay at
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Staphylococcus aureus (strain Mu50 / ATCC 700699)
Staphylococcus aureus (strain Mu50 / ATCC 700699)
Staphylococcus aureus (strain Mu50 / ATCC 700699)
Staphylococcus aureus (strain Mu50 / ATCC 700699)
Staphylococcus aureus (strain Mu50 / ATCC 700699)
Staphylococcus aureus (strain Mu50 / ATCC 700699)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
120000
-
gel filtration, glycerol density gradient sedimentation
132000
gel filtration, GatABC complex; gel filtration, GatABC complex; gel filtration, GatABC complex
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
-
2 * 63000, SDS-PAGE
heterotrimer
-
-
homodimer
Methanothermobacter thermautotrophicum
-
gel filtration, GatDE forms a homodimer with the binding interface between the D-subunits
trimer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
the glutamine transamidosome complex consisting of tRNAGln, glutamyltRNA synthase (GluRS) and the heterotrimeric amidotransferase GatCAB is crystalyzed at 3.35 A. Crystals are grown by the sitting-drop vapour-diffusion method at 20C
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
DEAE-cellulose column chromatography, Ni-NTA resin column chromatography, and Superdex 200 gel filtration
-
overexpressed with pTrcgatCABBST in E. coli
-
using heat treatment and column chromatography
using Ni-NTA chromatography
Methanothermobacter thermautotrophicum
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloning of the three genes, gatC, gatA, and gatB, which constitute the transcriptional unit of the enzyme
-
expressed in Escherichia coli as a His-tagged fusion protein
Methanothermobacter thermautotrophicum
-
expression in Escherichia coli
-
overexpressed in Escherichia coli
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
S128T
-
mutant protein retains significant glutaminase activity and transamidase activity in the presence of Gln
S152A
-
mutant is glutaminase inactive
S152T
-
mutant is glutaminase inactive
D178E
Methanothermobacter thermautotrophicum
-
glutamine hydrolysis is negligible, Gln-tRNAGln formation is undetectable
D178N
Methanothermobacter thermautotrophicum
-
glutamine hydrolysis is negligible, Gln-tRNAGln formation is undetectable
K254E
Methanothermobacter thermautotrophicum
-
glutamine hydrolysis is negligible, Gln-tRNAGln formation is undetectable
T101A
Methanothermobacter thermautotrophicum
-
glutamine hydrolysis is negligible, Gln-tRNAGln formation is undetectable
T101S
Methanothermobacter thermautotrophicum
-
hydrolyzes about 10% of glutamine compared to wild-type enzyme. Compared to wild-type enzyme, the mutant enzyme converts approximately half as much mischarged tRNA substrate to product
T177S
Methanothermobacter thermautotrophicum
-
mutant enzyme hydrolyzes the same amount of glutamine as the wild-type enzyme. As the wild-type enzyme, the mutant enzyme transforms most of Glu-tRNAGln to Gln-tRNAGln
T177V
Methanothermobacter thermautotrophicum
-
glutamine hydrolysis is negligible. Gln-tRNAGln formation is undetectable
additional information
Methanothermobacter thermautotrophicum
-
activity of the engineered enzyme variants indicates that the acceptor stem loop is the principle discrimination element because insertion of this loop alone enhances the specificity of the archaeal enzyme toward tRNAGln2
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
-
simple system for monitoring the inhibition of GatCAB activity using Escherichia coli Top10 co-expressing the non-discriminating glutamyl-tRNA synthetase ndGluRS and GatCAB genes from Staphylococcus aureus Mu50. Growth repression is confirmed by introducing ndgluRS from Staphylococcus aureus Mu50 into Escherichia coli. Co-expression of the gatCAB operon alleviates growth repression in the host Escherichia coli. The screening system consists of these two transformants and non-expressing Escherichia coli Top10. The transformant harbors both ndGluRS gene and GatCAB operon can be co-expressed in the presence and in the absence of chemical compounds of interest. There is no inhibitor that inactivates GatCAB activity, but upon expression of two inactive GatCAB deletion variants, GatCAB-10 and GatCAB-CHD, together with ndGluRS in Escherichia coli Top10, the cells show repressed growth as well as ndGluRS is expressed
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