Information on EC 2.7.7.42 - [glutamine synthetase] adenylyltransferase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
2.7.7.42
-
RECOMMENDED NAME
GeneOntology No.
[glutamine synthetase] adenylyltransferase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + [glutamine synthetase]-L-tyrosine = diphosphate + [glutamine synthetase]-O4-(5'-adenylyl)-L-tyrosine
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
adenylylation
deadenylylation
nucleotidyl group transfer
-
-
-
-
SYSTEMATIC NAME
IUBMB Comments
ATP:[glutamine synthetase]-L-tyrosine adenylyltransferase
This bacterial enzyme adenylates a tyrosine residue of EC 6.3.1.2, glutamine synthetase. The enzyme is bifunctional, and also catalyses a reaction that removes the adenyl group from the modified tyrosine residue (cf. EC 2.7.7.89, [glutamine synthetase]-adenylyl-L-tyrosine phosphorylase) [7,8]. The two activities are present on separate domains.
CAS REGISTRY NUMBER
COMMENTARY hide
9077-66-1
-
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
adenylyl-[glutamine synthase] + phosphate
glutamine synthase + ADP
show the reaction diagram
ADP + glutamine synthetase
adenyl-[glutamine synthetase] + phosphate
show the reaction diagram
-
-
-
-
r
ATP + glutamine synthetase
adenylated glutamine synthetase + diphosphate
show the reaction diagram
ATP + glutamine synthetase
glutamine synthetase-AMP + diphosphate
show the reaction diagram
the enzyme has two activities, adenylyl removase and adenylyl transferase, which are located in distinct catalytic domains that are separated by a regulatory domain
-
-
?
ATP + [L-glutamate:ammonia ligase (ADP-forming)]
diphosphate + [L-glutamate:ammonia ligase (ADP-forming)] -(AMP)
show the reaction diagram
ATP + [L-glutamate:ammonia ligase (ADP-forming)]
diphosphate + [L-glutamate:ammonia ligase (ADP-forming)]-(AMP)
show the reaction diagram
glutamine synthase + ATP
adenylyl-[glutamine synthase] + diphosphate
show the reaction diagram
glutamine synthetase-AMP + phosphate
ADP + glutamine synthetase
show the reaction diagram
the enzyme has two activities, adenylyl removase and adenylyl transferase, which are located in distinct catalytic domains that are separated by a regulatory domain
-
-
?
[L-glutamate:ammonia ligase (ADP-forming)]-O4-(5'-adenylyl)-L-tyrosine + H2O
adenylate + [L-glutamate:ammonia ligase (ADP-forming)]-L-tyrosine
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
ADP + glutamine synthetase
adenyl-[glutamine synthetase] + phosphate
show the reaction diagram
-
-
-
-
r
ATP + glutamine synthetase
adenylated glutamine synthetase + diphosphate
show the reaction diagram
A7Y9V0
-
-
-
r
ATP + glutamine synthetase
glutamine synthetase-AMP + diphosphate
show the reaction diagram
P30870
the enzyme has two activities, adenylyl removase and adenylyl transferase, which are located in distinct catalytic domains that are separated by a regulatory domain
-
-
?
ATP + [L-glutamate:ammonia ligase (ADP-forming)]
diphosphate + [L-glutamate:ammonia ligase (ADP-forming)] -(AMP)
show the reaction diagram
glutamine synthase + ATP
adenylyl-[glutamine synthase] + diphosphate
show the reaction diagram
-
ATase primarily regulated by alpha-ketoglutarate, glutamine has no effect on neither the adenylylation nor the deadenylylation of glutamine synthetase, PII proteins only stimulate the adenylylation reaction
-
-
r
glutamine synthetase-AMP + phosphate
ADP + glutamine synthetase
show the reaction diagram
P30870
the enzyme has two activities, adenylyl removase and adenylyl transferase, which are located in distinct catalytic domains that are separated by a regulatory domain
-
-
?
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
K+
-
adenylyltransferase and adenylyl-removing assay with 10 mM KCl
K2HPO4
-
deadenylylation assay with 10mM K2HPO4
additional information
-
Ca2+, Zn2+ and Cu2+ at 10 mM are ineffective
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-oxoglutarate
3-phosphoglycerate
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66% inhibition at 20 mM
4-Methyl-L-glutamate
-
-
6-diazo-5-oxonorleucine
-
-
alpha-ketoglutarate
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in Rhodospirillum rubrum alpha-ketoglutarate inhibits the adenylylation activity of ATase
D-glutamine
-
-
diphosphate
DL-2-aminobutyric acid
-
-
glutamate
-
L- and D-isomer
glutamine
L-methionine
-
-
L-tryptophan
-
-
phosphate
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-
PII signal transduction protein
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adenylyl-removing activity
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PII-UMP
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adenylyltransferase activity
S-(2-Hydroxyethyl)-L-cysteine
-
-
signal transduction protein PII
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inactivates the adenylyl-removing reaction
signal transduction protein PII-UMP
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reaction is inhibited by signal transduction protein PII-UMP
sulfate
-
-
uridylated PII signal transduction protein
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inhibits the adenylyltransferase reaction
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uridylated signal transduction protein PII
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the adenylyltransferase reaction is activated by glutamine and by the unmodified form of the PII signal transduction protein and is inhibited by the uridylylated form of PII, PII-UMP. PII, PII-UMP, and glutamine shift the enzyme among at least six different enzyme forms, two of which are inactive, one of which exhibits adenylyl-removing activity, and three of which exhibit adenylyltranferase activity. The enzyme appears to contain two distinct sites for PII and PII-UMP. The PII, PII-UMP, and glutamine sites are in communication. The binding of PII is favored by glutamine and its level reduced by PII-UMP, whereas glutamine and PII-UMP compete for the enzyme
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additional information
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inactivation scheme
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-oxoglutarate
-
controls the extent of activation or inhibition of the enzyme by PII or PII-UMP. 2-oxoglutarate acts exclusively through its binding to PII and PII-UMP, and does not alter the binding of PII or PII-UMP to the enzyme
alpha-ketoglutarate
Gln
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C-terminal truncation constructs are dependent on Gln for full adenylylation activity; wild-tpye AT needs both signal transduction protein PII and Gln to stimulate full adenylylation activity
glutamine
L-glutamine
PII proteins
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PII proteins only stimulate the adenylylation activity of ATase
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PII regulatory protein
PII signal transduction protein
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PII-UMP
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adenylyl-removing activity
signal transduction protein PII
signal transduction protein PII-UMP
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activates the adenylyl-removing reaction, enzyme appears to have two distinct sites for signal transduction protein PII and signal transduction protein PII-UMP
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0009
adenylyl-[glutamine synthase]
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adenylyl-removing activity, 0.00002 mM ATase, 1 mM alpha-ketoglutarate, 0.0005 mM signal transduction protein PII-UMP, 1 mM ATP, 5 mM potassium phosphate
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0.15 - 1.42
ATP
0.0029 - 0.006
glutamine synthase
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0.0032
glutamine synthetase
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pH 7.5, 30°C
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3.9
MgATP2-
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pH 7.5, 22°C
0.33
potassium phosphate
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adenylyl-removing activity, 0.00005 mM ATase, 1 mM alpha-ketoglutarate, 0.0005 mM signal transduction protein PII-UMP, 1 mM ATP, 0.00034 GS-AMP mM
0.005 - 1.1
[L-glutamate:ammonia ligase (ADP-forming)]
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0008 - 90
signal transduction protein PII
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8 - 8.2
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adenylylation
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.5 - 9.8
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pH 5.5: about 50% of activity maximum, pH 9.8: about 35% of activity maximum
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.2
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isoelectric focusing
PDB
SCOP
CATH
ORGANISM
UNIPROT
Escherichia coli (strain K12)
Escherichia coli (strain K12)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
64000
-
low and high speed sedimentation equilibrium, 115000 MW enzyme form is slowly converted during storage at 4°C to a smaller protein that is active only in adenylylation, not in deadenylylation
114000
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1 * 114000, high speed sedimentation study of the enzyme in 6 M guanidine-HCl
115000
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ultracentrifugation
126200
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estimated from gene sequence
145000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
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1 * 114000, high speed sedimentation study of the enzyme in 6 M guanidine-HCl
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
hanging-drop vapour-diffusion method, C-terminal domain, residues 441-945
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N-terminal domain, AT-N440
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structure of the N-terminal domain, residues 1-440, containing the regulatory and the adenylyl transferase domains, to 2.4 A resolution. Deduction of a model of the complete enzyme and a model for the complex with glutamine synthetase and the nitrogen signal transduction protein PII; the structure of the C-terminal fragment, containing residues 449-946, of glutamine synthetase adenylyl transferase is determined to a resolution of 2.4 A
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3
-
4°C, 12 h, loss of 70% of initial activity
643329
4 - 9
-
4°C, 12 h, no loss of activity
643329
9.5
-
4°C, 12 h, loss of 50% of initial activity
643329
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
bovine serum albumin, above 1 mg/ml, prevents inactivation at 4°C and 25°C and aggregation
-
considerably less stable in Tris or imidazole buffer than in a magnesium phosphate buffer
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Mg2+, 20 mM protects to some extent against heat inactivation
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no stabilization by ATP, CTP, Mn2+, glutamine, cysteine or mercaptoethanol each at 20 mM, 2 mM DTT, 20% glycerol, sucrose, polyethyleneglycol or urea at 1 M
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-80°C, purified enzyme, stored after quick freezing with liquid N2, potassium phosphate buffer, 10-100 mM, pH 7.6, 1 mM MgCl2, stable for months at enzyme concentration above 0.1 mg/ml
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0°C-4°C, enzyme concentration above 1 mg/ml, stable for 12 days
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4°C, enzyme solution of 3 mg/ml, loss of 50% activity within 30 days, the activity declines faster with more diluted enzyme solutions
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
2200fold to homogeneity
-
300fold to homogeneity
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ammonium sulfate fractionation, DE-52 column chromatography, Bio-Gel A0.5M gel filtration, and phenyl Sepharose column chromatography
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ATase is cloned into the pET101/D-TOPO plasmid, containing ATase fused to a C-terminal V5 epitope followed by six histidines, His-tagged proteins are applied to HisTrap HP 1ml column
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partial
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloned in Escherichia coli
cloned in Escherichia coli DH5alpha, TG1 and JM109
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overexpression
-
the plasmids pGEM-T Easy, pSUP202 and pRK2073 are used
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
gene expression is slightly repressed under nitrogen-excess conditions, and the repression is more pronounced under excess nitrogen plus carbon-limiting conditions. Variations in the concentration of uridylyltransferase and adenylyltransferase also affect the rate of glutamine synthetase synthesis
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gene GlnE is cotranscribed with another gene, orfXE. All three Gln regulatory genes, uridylyl-transferase (GlnD), the PII protein (GlnB), and adenyly I-transferase (gInE) are constitutively expressed at a low level, i.e. their expression is independent of the nitrogen status and the RNA polymerase sigma factor
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D173N/D175N
-
inactivation of the N-terminal nucleotidyltransferase domain by mutation of both highly conserved aspartates, mutated enzyme shows adenylyltransferase activity but lacks adenylyl-removing activity
D463N/P467A/L469G
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clustered point mutations in the central region of ATase, only 30-50% adenylyl-removing and adenylyltransferase activity of wild type enzyme, adenylyl-removing activity is inhibited by signal transduction protein PII and glutamine, adenylyltransferase activity is regulated by glutamine, signal transduction protein PII and signal transduction protein PII-UMP
D701N/D703N
-
inactivation of the C-terminal nucleotidyltransferase domain by mutation of both highly conserved aspartates, mutated enzyme shows adenylyl-removing activity but lacks adenylyltransferase activity
delata456-577
-
enzyme missing amino acids 456-577 from the central region of ATase lacks adenylyl-removing activity but retains adenylyltransferase activity, adenylyltransferase activity is inhibited by signal transduction protein PII and signal transduction protein PII-UMP
L525G/R527A/I528G
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clustered point mutations in the central region of ATase, enzyme completely lacks adenylyl-removing activity but retains adenylyltransferase activity, adenylyltransferase activity is strongly enhanced by glutamine
R499A/R501A/D505N/P509A/L511G
-
clustered point mutations in the central region of ATase, enzyme completely lacks adenylyl-removing activity but retains adenylyltransferase activity, enzyme is inhibited by either signal transduction protein PII or signal transduction protein PII-UMP, adenylyltransferase activity is strongly enhanced by glutamine
R571A/P573A/L575G
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clustered point mutations in the central region of ATase, enzyme shows approx. 12.5% of adenylyl-removing activity of wild-type enzyme, adenylyl-removing activity is inhibited by glutamine and signal transduction protein PII, adenylyltransferase activity is strongly activated by glutamine
D438Y/R657S/S684K
-
temperature-sensitive mutant
D720A
-
mutation in the adenylylation domain, compromises activity. Strain shows reduced growth rates in the low-ammonia- and glutamine-containing media
D732A
-
mutation in the adenylylation domain, mutation does not completely abrogate the enzyme activity
V921Q
-
temperature-sensitive mutant
D438Y/R657S/S684K
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temperature-sensitive mutant
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D720A
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mutation in the adenylylation domain, compromises activity. Strain shows reduced growth rates in the low-ammonia- and glutamine-containing media
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D732A
-
mutation in the adenylylation domain, mutation does not completely abrogate the enzyme activity
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V921Q
-
temperature-sensitive mutant
-
additional information
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