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
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain YMC10
-
-
Manually annotated by BRENDA team
strain AF2122/97
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-
Manually annotated by BRENDA team
strain AF2122/97
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-
Manually annotated by BRENDA team
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-
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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
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
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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
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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
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-
6-diazo-5-oxonorleucine
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-
alpha-ketoglutarate
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in Rhodospirillum rubrum alpha-ketoglutarate inhibits the adenylylation activity of ATase
D-glutamine
-
-
diphosphate
DL-2-aminobutyric acid
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-
glutamate
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L- and D-isomer
glutamine
L-methionine
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-
L-tryptophan
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-
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
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-
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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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temperature-sensitive mutant
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
D732A
-
mutation in the adenylylation domain, mutation does not completely abrogate the enzyme activity
V921Q
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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
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mutation in the adenylylation domain, mutation does not completely abrogate the enzyme activity
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V921Q
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temperature-sensitive mutant
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additional information
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