Information on EC 2.6.1.16 - glutamine-fructose-6-phosphate transaminase (isomerizing)

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

EC NUMBER
COMMENTARY hide
2.6.1.16
-
RECOMMENDED NAME
GeneOntology No.
glutamine-fructose-6-phosphate transaminase (isomerizing)
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
L-glutamine + D-fructose 6-phosphate = L-glutamate + D-glucosamine 6-phosphate
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
amino group transfer
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Alanine, aspartate and glutamate metabolism
-
-
Amino sugar and nucleotide sugar metabolism
-
-
Biosynthesis of antibiotics
-
-
CMP-legionaminate biosynthesis I
-
-
Metabolic pathways
-
-
UDP-N-acetyl-D-glucosamine biosynthesis I
-
-
UDP-N-acetyl-D-glucosamine biosynthesis II
-
-
UDP-GlcNAc biosynthesis
-
-
SYSTEMATIC NAME
IUBMB Comments
L-glutamine:D-fructose-6-phosphate isomerase (deaminating)
Although the overall reaction is that of a transferase, the mechanism involves the formation of ketimine between fructose 6-phosphate and a 6-amino group from a lysine residue at the active site, which is subsequently displaced by ammonia (transamidination).
CAS REGISTRY NUMBER
COMMENTARY hide
9030-45-9
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
Helminthosporium sativum
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
strain V1-1 (ATCC50447)
UniProt
Manually annotated by BRENDA team
strain V1-1 (ATCC50447)
UniProt
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
D-fructose 6-phosphate
D-glucose 6-phosphate
show the reaction diagram
L-gamma-glutamyl-p-nitroanilide + D-fructose 6-phosphate
?
show the reaction diagram
-
-
-
-
?
L-gamma-glutamyl-p-nitroanilide + H2O
L-glutamine + p-nitroaniline
show the reaction diagram
L-glutamine + D-fructose 6-phosphate
L-glutamate + D-glucosamine 6-phosphate
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
L-glutamine + D-fructose 6-phosphate
L-glutamate + D-glucosamine 6-phosphate
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
-
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(2S)-2-amino-3-(([(2R,3R)-3-benzoyloxiran-2-yl]carbonyl)amino)propanoic acid
-
-
(2S)-2-amino-3-([(2E)-4-oxo-4-phenylbut-2-enoyl]amino)propanoic acid
-
-
(2S)-2-amino-3-([(2E)-4-oxopent-2-enoyl]amino)propanoic acid
-
-
(2S)-3-(([(2R,3R)-3-acetyloxiran-2-yl]carbonyl)amino)-2-aminopropanoic acid
-
-
1,1'-dithiodiformamidine
1,1'-[1,3,4-thiadiazole-2,5-diylbis[sulfanediyl(1-oxoethane-2,1-diyl)]]ditetrahydropyridazine-3,6-dione
-
-
1,2-anhydrohexitol 6-phosphate
-
mixture of the four diastereoisomers. Irreversible inactivation. D-fructose 6-phosphate and 2-amino-2-deoxyglucitol protect, L-glutamine does not
1-methyl 8-(2-oxopropyl) (2E,7S)-7-amino-4-oxooct-2-enedioate
-
ester derivative of N3-(4-metoxyfumaroyl)-(S)-2,3-diaminopropanoic acid, potent inhibitory activity against fungal glucosamine-6-phosphate synthase, good antifungal activity against Candida albicans
1-methyl 8-[(2R)-3-oxobutan-2-yl] (2E,7S)-7-amino-4-oxooct-2-enedioate
-
ester derivative of N3-(4-metoxyfumaroyl)-(S)-2,3-diaminopropanoic acid, potent inhibitory activity against fungal glucosamine-6-phosphate synthase, good antifungal activity against Candida albicans
2,2'-(1,3,4-thiadiazole-2,5-diyldisulfanediyl)bis[N-(pyrrolidin-1-yl)acetamide]
-
-
2-amino-2-deoxy-D-glucitol 6-phosphate
2-amino-2-deoxy-D-glucitol-6-phosphate
2-amino-2-deoxy-D-glucitol-6-phosphate dimethyl ester
-
-
2-amino-2-deoxy-D-mannitol 6-phosphate
2-amino-2-deoxy-D-mannitol-6-phosphate
2-Amino-2-deoxyglucitol 6-phosphate
-
competitive with respect to D-fructose 6-phosphate
4,4'-dithiodipyridine
-
inactivation reversed by dithiothreitol. Competitive with respect to L-glutamine. Non-competitive with respect to D-fructose 6-phosphate
4-(furan-2-ylcarbonyl)-3-hydroxy-5-(4-phenoxyphenyl)-1-(pyridin-3-ylmethyl)-1,5-dihydro-2H-pyrrol-2-one
-
20% inhibition at 0.1 mM
5,5'-dithionitrobenzoic acid
-
5-phospho-D-arabinoamide
-
-
6,6'-Dithiodinicotinic acid
6,7-bis(2-methoxyphenyl)-10-methyl-1,4,7,12-tetrahydro-6H-chromeno[4,3-d][1,2,4]triazolo[1,5-a]pyrimidine
-
70% inhibition at 0.1 mM
6-diazo-5-oxo-L-norleucine
8-(3,3-dimethyl-2-oxobutyl) 1-methyl (2E,7S)-7-amino-4-oxooct-2-enedioate
-
ester derivative of N3-(4-metoxyfumaroyl)-(S)-2,3-diaminopropanoic acid, potent inhibitory activity against fungal glucosamine-6-phosphate synthase, good antifungal activity against Candida albicans
Aaptamine
-
IC50: 0.12 mM
amitrole
-
IC50: 0.1 mM
anticapsin
arabinose oxime 5-phosphate
-
inhibitor of the sugar isomerising domain
azaserine
D-glucitol 6-phosphate
-
competitive with respect to D-fructose 6-phosphate
D-Glucosamine 6-phosphate
-
negative feedback-regulation at post-transcriptional level. The biological function of small RNA GlmZ is to positively control the enzyme's mRNA in response to D-glucosamine 6-phosphate concentrations. YhbJ, a gene of the rpoN operon, negatively regulates GlmZ
D-Glucosamine-6-phosphate
-
1 mM, about 50% loss of activity
dihydroxyacetone
-
weak
dihydroxyacetone phosphate
-
weak
DL-delta-1-pyrroline-5-carboxylate
-
competitive with respect to L-glutamine
ethyl 2-[2-(3-bromophenyl)-3-[(4-fluorophenyl)carbonyl]-4-hydroxy-5-oxo-2,5-dihydro-1H-pyrrol 1-yl]-4-methyl-1,3-thiazole-5-carboxylate
-
70% inhibition at 0.1 mM
ethyl 2-[3-[(4-fluorophenyl)carbonyl]-4-hydroxy-2-(4-methoxyphenyl)-5-oxo-2,5-dihydro-1H pyrrol-1-yl]-4-methyl-1,3-thiazole-5-carboxylate
-
70% inhibition at 0.1 mM
fructose 1,6-diphosphate
-
weak
glyceraldehyde 3-phosphate
-
50% inhibition at 0.2 mM
glycolaldehyde
-
weak
Glyoxal
-
50% inhibition at 0.03 mM
iodoacetamide
L-2,3-diaminopropanoic acid
L-alpha-glycerophosphate
-
weak
Mercuric chloride
-
84% inhibition at 1 mM
methyl (2E)-4-([(2S)-2,3-diamino-3-oxopropyl]amino)-4-oxobut-2-enoate
-
-
methyl (2E)-4-([(2S)-2-amino-3-(methylamino)-3-oxopropyl]amino)-4-oxobut-2-enoate
-
-
methylglyoxal
N-acetyl-2-amino-2-deoxy-D-glucitol-6-phosphate
-
-
N-ethylmaleimide
N-iodoacetylglucosamine 6-phosphate
-
D-fructose 6-phosphate protects
N3-(4-Methoxyfumaroyl)-L-2,3-diaminopropanoic acid
N3-bromoacetyl-L-2,3-diaminopropanoic acid
N3-chloroacetyl-L-2,3-diaminopropanoic acid
N3-fumaramoyl-L-2,3-diaminopropanoic acid
N3-Fumaroyl-L-2,3-diaminopropanoic acid
N3-Fumaroylcarboxyamido-L-2,3-diaminopropionic acid
-
N3-iodoacetyl-L-2,3-diaminopropanoic acid
N3-L-trans-epoxysuccinamoyl-L-2,3-diaminopropanoic acid
-
inhibitor of the glutamine binding site
N4-(4-Methoxyfumaroyl)-L-2,4-diaminobutanoic acid
-
-
p-chloromercuribenzoate
pyridoxamine-5'-phosphate
-
-
Tolbutamide
-
80% inhibition at 2 mg/ml
UDP-glucose
-
-
UDP-N-acetyl-alpha-D-glucosamine
-
-
UDP-N-acetylglucosamine
uridine 5'-diphospho-N-acetyl-D-glucosamine
-
-
uridine 5'-diphospho-N-acetylglucosamine
-
additional information
-
no inhibition by 0.1 mM c3, 4, 6, 9, 12, 13
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
chondroitin sulfate B
-
-
desferrioxamine
-
0.2 mM; activation of gene expression via iron chelation
dithiothreitol
-
1 mM, activates 1.8fold
Picolinic acid
-
2 mM; activation of gene expression via iron chelation
additional information
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.2 - 6.5
D-fructose 6-phosphate
16
D-glucose 6-phosphate
-
pH 7.2, 37°C, isomerization catalyzed by C-terminal domain
1
glutamine
-
-
0.67
L-gamma -glutamyl-p-nitroanilide
-
reaction mixture contains 1 mM L-gamma-glutamyl-p-nitroanilide, 1 mM EDTA, 1 mM DTT and the appropriately diluted enzyme preparation in 20 mM HEPES buffer (pH 7.5) at 25°C
0.0064 - 12.2
L-gamma-glutamyl-p-nitroanilide
0.2 - 2.36
L-glutamate
0.04 - 5.1
L-glutamine
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.004 - 35.5
D-fructose 6-phosphate
0.00092 - 0.68
L-gamma-glutamyl-p-nitroanilide
9.81 - 12
L-glutamate
0.28 - 35.5
L-glutamine
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.45
(2S)-2-amino-3-(([(2R,3R)-3-benzoyloxiran-2-yl]carbonyl)amino)propanoic acid
-
pH 7.0, 37°C
0.056
(2S)-2-amino-3-([(2E)-4-oxo-4-phenylbut-2-enoyl]amino)propanoic acid
-
pH 7.0, 37°C
15.2
(2S)-2-amino-3-([(2E)-4-oxopent-2-enoyl]amino)propanoic acid
-
pH 7.0, 37°C
19.4
(2S)-3-(([(2R,3R)-3-acetyloxiran-2-yl]carbonyl)amino)-2-aminopropanoic acid
-
pH 7.0, 37°C
0.018
2-amino-2-deoxy-D-glucitol 6-phosphate
-
pH 7.5, 37°C
0.035
2-amino-2-deoxy-D-glucitol-6-phosphate
-
-
0.68
2-amino-2-deoxy-D-glucitol-6-phosphate dimethyl ester
-
-
0.00067
2-amino-2-deoxy-D-mannitol 6-phosphate
-
pH 7.5, 37°C
0.009
2-amino-2-deoxy-D-mannitol-6-phosphate
-
-
0.0193 - 0.088
2-Amino-2-deoxyglucitol 6-phosphate
0.095
4-Glutamylhydroxamate
-
pH 7.5, 37°C
0.62
5-phospho-D-arabinoamide
-
-
0.001 - 0.0075
6-diazo-5-oxo-L-norleucine
0.00011 - 0.00075
anticapsin
2.46
D-glucitol 6-phosphate
-
pH 7.2
0.023
DL-delta-1-pyrroline-5-carboxylate
-
-
0.56
Glucosamine-6-phosphate
-
wild type enzyme
12.6
iodoacetamide
-
pH 7.5, room temperature
0.84
N-acetyl-2-amino-2-deoxy-D-glucitol-6-phosphate
-
-
0.00027 - 0.00035
N3-(4-Methoxyfumaroyl)-L-2,3-diaminopropanoic acid
0.0001
N3-bromoacetyl-L-2,3-diaminopropanoic acid
-
pH 6.5, 25°C
0.0006
N3-chloroacetyl-L-2,3-diaminopropanoic acid
-
pH 6.5, 25°C
0.019
N3-fumaramoyl-L-2,3-diaminopropanoic acid
-
pH 7.5, 37°C
0.008 - 0.0551
N3-Fumaroyl-L-2,3-diaminopropanoic acid
0.125
N3-Fumaroylcarboxyamido-L-2,3-diaminopropionic acid
-
pH 7.5, 37°C
-
0.000125
N3-iodoacetyl-L-2,3-diaminopropanoic acid
-
pH 6.5, 25°C
0.0013 - 0.04
UDP-N-acetylglucosamine
additional information
anticapsin
-
KI varying from 0.001 - 1
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.66
(2S)-2-amino-3-(([(2R,3R)-3-benzoyloxiran-2-yl]carbonyl)amino)propanoic acid
Saccharomyces cerevisiae
-
pH 7.0, 37°C
0.27
(2S)-2-amino-3-([(2E)-4-oxo-4-phenylbut-2-enoyl]amino)propanoic acid
Saccharomyces cerevisiae
-
pH 7.0, 37°C
5.6
(2S)-2-amino-3-([(2E)-4-oxopent-2-enoyl]amino)propanoic acid
Saccharomyces cerevisiae
-
pH 7.0, 37°C
7.3
(2S)-3-(([(2R,3R)-3-acetyloxiran-2-yl]carbonyl)amino)-2-aminopropanoic acid
Saccharomyces cerevisiae
-
pH 7.0, 37°C
0.0051
1-methyl 8-(2-oxopropyl) (2E,7S)-7-amino-4-oxooct-2-enedioate
Candida albicans
-
pH not specified in the publication, temperature not specified in the publication
0.0049
1-methyl 8-[(2R)-3-oxobutan-2-yl] (2E,7S)-7-amino-4-oxooct-2-enedioate
Candida albicans
-
pH not specified in the publication, temperature not specified in the publication
0.056 - 0.085
2-amino-2-deoxy-D-glucitol-6-phosphate
0.073
2-amino-2-deoxy-D-mannitol 6-phosphate
Sporothrix schenckii
D3YIG6
pH not specified in the publication, temperature not specified in the publication
0.0052
8-(3,3-dimethyl-2-oxobutyl) 1-methyl (2E,7S)-7-amino-4-oxooct-2-enedioate
Candida albicans
-
pH not specified in the publication, temperature not specified in the publication
0.12
Aaptamine
Escherichia coli
-
IC50: 0.12 mM
0.1
amitrole
Escherichia coli
-
IC50: 0.1 mM
0.081
methyl (2E)-4-([(2S)-2,3-diamino-3-oxopropyl]amino)-4-oxobut-2-enoate
Candida albicans
-
pH 7.0, 37°C
0.127
methyl (2E)-4-([(2S)-2-amino-3-(methylamino)-3-oxopropyl]amino)-4-oxobut-2-enoate
Candida albicans
-
pH 7.0, 37°C
0.013 - 0.12
N3-(4-Methoxyfumaroyl)-L-2,3-diaminopropanoic acid
0.52
UDP-N-acetyl-alpha-D-glucosamine
Saccharomyces cerevisiae
-
pH 7.0, 37°C
0.45
UDP-N-acetylglucosamine
Sporothrix schenckii
D3YIG6
crude enzyme preparation, pH not specified in the publication, temperature not specified in the publication
1.55 - 2
uridine 5'-diphospho-N-acetyl-D-glucosamine
0.16
uridine 5'-diphospho-N-acetylglucosamine
Volvariella volvacea
Q6DLZ8
recombinant enzyme, in 10 mM KCl, 1 mg/ml bovine serum albumin, 20 mM imidazole buffer (pH 6.8), 1 mM EDTA, 1 mM dithiothreitol, and 10% (v/v) glycerol
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.001
-
mutant enzyme L468P
0.0047
-
-
0.021
-
glutamine 6-phosphate-synthetic activity, mutant V711F, pH 7.5, 25°C
0.05
-
glutamine 6-phosphate-synthetic activity, mutant W97G, pH 7.5, 25°C
0.095
-
amidohydrolysing activity of the mutant enzyme W97G using D-fructose 6-phosphate as substrate
0.256
-
amidohydrolysing activity of the mutant enzyme W97F using D-fructose 6-phosphate as substrate
0.38
-
mutant enzyme I271T; mutant enzyme I3T; mutant enzyme S449P
0.39
-
wild type enzyme
0.42
-
mutant enzyme A38T; mutant enzyme G471S
0.631
-
wild type enzyme, using D-fructose 6-phosphate as a substrate
0.89
-
amidohydrolysing activity of the wild type enzyme using D-fructose 6-phosphate as substrate
0.94
-
amidohydrolysing activity of the mutant enzyme V711F using D-fructose 6-phosphate as substrate
12
-
glutamine 6-phosphate-synthetic activity, wild-type, pH 7.5, 25°C
34.8
-
-
additional information
-
development of an assay for glucosamine 6-phosphate synthase that measures the production of glucosamine 6-phosphate by either following the consumption of acetyl-CoA spectrophotometrically at 230 nm or quantifying the free thiol with 5,5'-dithio-bis(2-nitrobenzoic acid), i.e. Ellman’s reagent in a discontinuous manner. Simple assay method, which can be adapted to 96-well microtiter plate format
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.1 - 6.4
-
-
6.2 - 6.7
-
potassium phosphate buffer
6.5 - 7.5
-
-
7
-
potassium phosphate buffer
7.4
-
-
7.7
-
assay at
7.7 - 7.9
-
-
7.7
-
assay at
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 8
marked decrease in activity at pH values below 6.0 and above 8.0
6 - 8.5
-
-
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
mononuclear phagocyte, constitutive expression of glutamine:fructose 6-phosphate amidotransferase mRNA and protein. Induction of expression by picolinic acid, hypoxia and desferrioxamine
Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
UNIPROT
Candida albicans (strain SC5314 / ATCC MYA-2876)
Candida albicans (strain SC5314 / ATCC MYA-2876)
Candida albicans (strain SC5314 / ATCC MYA-2876)
Candida albicans (strain SC5314 / ATCC MYA-2876)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Francisella tularensis subsp. tularensis (strain SCHU S4 / Schu 4)
Shewanella denitrificans (strain OS217 / ATCC BAA-1090 / DSM 15013)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
39500
-
4 * 39500, hexose phosphate-isomerizing domain, Superdex 200 HR 10/30 gel filtration
41000
-
glutamine amide-hydrolysing domain, SDS-PAGE
66640
-
SDS-PAGE
68600
x * 68600, calculated for native protein, x * 69600, calculated, and x * 69400, SDS-PAGE, His-tagged protein, respectively
69400
x * 68600, calculated for native protein, x * 69600, calculated, and x * 69400, SDS-PAGE, His-tagged protein, respectively
69600
x * 68600, calculated for native protein, x * 69600, calculated, and x * 69400, SDS-PAGE, His-tagged protein, respectively
70000
-
2 * 70000, calculated by means of crystalline structure determined by X ray diffraction
70800
-
2 * 70800, SDS-PAGE
75000
-
4 * 75000, gel filtration, SDS-PAGE
79000
-
SDS-PAGE, wild-type and mutants
79140
-
Superdex 200 HR 10/30 gel filtration
159000
-
hexose phosphate-isomerizing domain, Superdex 200 HR 10/30 gel filtration
160000
-
gel filtration, mutant Gfa1-His6DELTA655-660
170000 - 193000
-
gel filtration
280000
-
gel filtration
300000
-
ultrafiltration
306000
estimated from amino acid sequence
310000
gel filtration
320000
-
gel filtration
328000
-
gel filtration, mutant Gfa1-K568H/S569H
335000
-
gel filtration, mutant Gfa1-His6DELTA343-348
340000
350000
410000
-
gel filtration, untreated enzyme
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hexamer
-
the enzyme is regulated by a morpheein-type allosteric mechanism, in which functional dimeric GlmS is in equilibrium with the inactive hexamer
homotetramer
monomer
-
1 * 42000, glutamine amide-hydrolysing domain, Superdex 200 HR 10/30 gel filtration
oligomer
-
sedimentation velocity experiments show that at low concentration the enzyme is mainly present as a dimer. At a higher protein concentration the equilibrium between the two forms of GlmS is significantly displaced toward the oligomeric form
tetramer
additional information
-
The fact that the 77 kDa protein expressed in Escherichia coli is sensitive to inhibition by UDP-N-acetylglucosamine is consistent with the idea that mammalian GFAT is comprised of four identical subunits
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
phosphoprotein
-
enzyme is phosphorylated in vivo, phosphorylation site is residue S243. Phosphorylation at Ser243 stimulates glucosamine 6-phosphate synthesizing activity, lowers amidohydrolyzing activity in the absence of fructose 6-phosphate, and lowers Km of fructose 6-phosphate 2fold, but has no effect on UDP-GlcNAc inhibition. Phosphorylation is mediated by AMP-activated protein kinase and calcium/calmodulin-dependent kinase II in vitro
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystals are composed of a trans-acting form of the Thermoanaerobacter tengcongensis ribozyme with a single 2'-deoxyribose substitution at the cleavage site, which traps the RNA in the precleavage state. These crystals yield an accurate structure of the RNA to 1.7 A resolution
-
crystal structure of the isomerase domain in complex with the allosteric inhibitor UDP-GlcNAc and in the presence of glucose 6-phosphate, fructose 6-phosphate and an analogue of the reaction intermediate, 2-amino-2-deoxy-D-mannitol 6-phosphate. Deduction of a solution structure of the native protein. The tetrameric enzyme can be described as a dimer of dimers, with each half similar to the related enzyme from Escherichia coli. The core of the protein consists of the isomerase domains
hanging drop vapour diffusion and sitting drop vapour diffusion in 20% (w/v) PEG 600 and 0.15 M KSCN at pH 8.5
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molecular docking of inhibitors methyl (2E)-4-([(2S)-2,3-diamino-3-oxopropyl]amino)-4-oxobut-2-enoate and methyl (2E)-4-([(2S)-2-amino-3-(methylamino)-3-oxopropyl]amino)-4-oxobut-2-enoate
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crystal structure of intact protein
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crystal structures of enzyme alone and in complex with the glucosamine 6-phosphate product at 2.95 A and 2.9 A resolution, respectively. No electron density for the glutaminase domain is observed. Upon sugar binding, the C-terminal loop, which forms the major part of the channel walls, becomes ordered and covers the synthase site. The ordering of the glutaminase domains likely follows fructose 6-phosphate binding by the anchoring of Trp74, which acts as the gate of the channel, on the closed C-terminal loop. This is accompanied by a major conformational change of the side chain of Lys503 of the neighboring synthase domain that strengthens the interactions of the synthase domain with the C-terminal loop and completely shields the synthase site. The concomitant conformational change of theLys503-Gly505 tripeptide places catalytic His504 in the proper position to open the sugar and buries the linear sugar, which is now in the vicinity of the catalytic groups involved in the sugar isomerization reaction
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docking study using inhibitors 1,1'-[1,3,4-thiadiazole-2,5-diylbis[sulfanediyl(1-oxoethane-2,1-diyl)]]ditetrahydropyridazine-3,6-dione, 2,2'-(1,3,4-thiadiazole-2,5-diyldisulfanediyl)bis[N-(pyrrolidin-1-yl)acetamide]. The binding pocket of the enzyme includes the residues, Cys300, Gly301, Thr302, Ser303, Ser347, Gln348, Ser349, Thr352, Val399, Ser401, Ala602 and Lys603. The high docking energies of all generated conformers of 1,1'-[1,3,4-thiadiazole-2,5-diylbis[sulfanediyl(1-oxoethane-2,1-diyl)]]ditetrahydropyridazine-3,6-dione are strongly proportional to the antibacterial activities
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hanging drop vapour diffusion method, 12% polyethylene glycol 8000, 0.1 M KCl, 5% glycerol
molecular dynamics simulations. Key role for Trp74, in the sealing of the hydrophobic channel connecting the two binding sites, as well as for the two Ala602 and Val605 residues, which form a narrow passage whose opening/closing constitutes an essential event in ammonia transfer
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the crystal structure of the C1A mutant of Escherichia coli GlmS, solved at 2.5 A resolution, is organized as a hexamer, where the glutaminase domains adopt an inactive conformation
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two crystal complexes of the isomerase domain with D-glucose 6-phosphate and 2-amino-2-deoxyglucitol 6-phosphate
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isomerase domain of the human GFAT in the presence of cyclic glucose-6-phosphate and linear D-glucosamine 6-phosphate, hanging drop vapor diffusion method, at 20°C using 12% (v/v) isopropanol, 0.8 M ammonium acetate and 40 mM Tris-HCl at pH 8.5
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molecular model of the 3-D on the basis of the crystal structures of Escherichia coli GlcN-6-P synthase, PDB entry 4AMV and Candida albicans GlcN-6-P synthase, PDB entry 2POC. The protein has a terminal glutamine amidotransferase type 2 domain (residues 2-318). The phosphosugar-binding SIS domain (residues 390-529; 562-707) plays a key role in catalysis and communication of fructose 6-phosphate and D-glucosamine 6-phosphate between the two active sites
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.5 - 5.5
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737920
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
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90 min, complete loss of activity without stabilizing agent. Dithiothreitol, D-fructose 6-phosphate, D-glucose 6-phosphate or L-glutamine, 1 mM, increase thermal stability at 37°C
45
half-life of 1 h at 45°C
50
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activity of the wild type enzyme is not affected by incubation at 50°C for 60 min
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
can be concentrated by precipitation with 2.3 M ammonium sulfate with some concomitant loss of activity
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complete loss of activity after treatment with ammonium sulfate, 30-50% saturation
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dithiothreitol, D-fructose 6-phosphate, D-glucose 6-phosphate or L-glutamine, 1 mM, increases thermal stability at 37°C
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extremely unstable during purification
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freezing and thawing has no effect on activity
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glucose 6-phosphate, 12 mM, required as protective agent during purification
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inactivated by freezing and thawing
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inactivated when exposed to a solution of pH 5.5 or below
isopropanol, 1%, stabilizes during purification
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sucrose needed as stabilizing agent
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very unstable
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-18°C, little loss of activity after 1 week for lyophilized material after calcium phosphate gel treatment
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-20°C, 0.6 M sucrose, 60% loss of activity after 45 days
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-20°C, potassium phosphate buffer, pH 7.0, containing 1 mM EDTA, 1 mM DTT, 50% glycerol and 10 mM fructose 6-phosphate, fully stable
-25°C, 1 month, 15% activity loss, ammonium sulfate precipitated enzyme
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-80°C, 3 months
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-80°C, 50 mM Tris-HCl, pH 7.8, 200 mM NaCl, 1 mM fructose 6-phosphate, 2 mM tris(2-carboxyethyl)phosphine hydrochloride, 200 mM imidazole, 10% glycerol, stable for up to 12 months
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0-2°C, potassium phosphate buffer, pH 7.0, 0.06 M L-glutamine, 0.01 M EDTA, stable for up to 3 or 4 weeks
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4°C or -15°C, 8 days, 50% activity loss
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4°C or 15°C, 50% loss of activity after 8 days
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4°C, 25 mM potassium phosphate buffer, pH 7.5, 1 mM EGTA, 2 mM dithiothreitol, 500 mM sucrose, stable for up to 2 or 3 weeks
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4°C, 50 mM MOPS (pH 6.5), 1 mM dithiothreitol, 1 mM EDTA, and 10 mM KCl, overnight storage, recombinant enzyme activity is low and no longer evident with preparations losing 60% of their activity
4°C, potassium phosphate buffer, pH 7.0, containing 1 mM EDTA, 1 mM DTT, 50% glycerol and 10 mM fructose 6-phosphate, complete loss of activity within 24 h
5°C, potassium phosphate buffer, pH 7.5, 1 mM EDTA, 1 mM dithiothreitol, 0.5 mM L-glutamine, 0.05 mM D-glucose 6-phosphate, 600 mM sucrose, stable for few weeks
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in liquid N2, stable for up to 2 weeks
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Bio-Spin 6 column chromatography
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enzyme is purified to at least 98% homogeneity with 42% final yield
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Ni-NTA column chromatography
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Ni2+-IDA agarose affinity chromatography and HiTrap desalting column chromatography
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Ni2+-IDA agarose chromatography
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Ni2+-IDA agarose chromatography and Superdex 200 gel filtration
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partial purification by immobilized Ni2+ affinity column chromatography
Q-Sepharose fast flow column chromatography and Superdex 200 Hiload gel filtration
Q-Sepharose fast flow column chromatography and Superdex 200 HR 26/60 Hiload gel filtration
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recombinant protein
using one-step immobilized metal-ion affinity chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli
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expressed in Escherichia coli BL21(DE3) cells
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expressed in Escherichia coli BL21(DE3) pLysS cells
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expressed in Escherichia coli BL21(DE3)pLysS cells
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expressed in Escherichia coli JM109 cells
expressed in Escherichia coli strain BL21(DE3) pLysS
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expressed in Saccharomyces cerevisiae strain YRS C-65
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expression as His-tagged protein
expression in insect cell and Escherichia coli
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expression in Pichia pastoris
expression in Saccharomyces cerevisiae
expressiuon in Escherichia coli
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overexpressed in Escherichia coli
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
G40A
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mutant with exchanged guanine is inactive. The 2.7 A resolution crystal structure of the mutant shows that the RNA is in a conformation nearly identical to that of the wild-type glmS ribozyme. The experimental electron density maps indicate that GlcN6P binds to the G40A mutant in the same location as in the wild-type ribozyme. Raman pH titrations of GlcN6P using crystals of the G40A mutant glmS ribozyme show that the pKa of the amine of the ribozyme-bound GlcN6P differs substantially for the wild-type and G40A mutant ribozymes
V711F
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mutation results in an almost complete elimination of the GlcN-6-P-synthetic activity, with the retention of the amidohydrolase and sugar phosphate-isomerizing activities; reduction of the glucosamine 6-phosphate-synthetic activity, with the retention of the amidohydrolase and sugar phosphate-isomerizing activities
W97F
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mutation results in an almost complete elimination of the GlcN-6-P synthetic activity, with the retention of the amidohydrolase and sugar phosphate-isomerizing activities; reduction of the glucosamine 6-phosphate-synthetic activity, with the retention of the amidohydrolase and sugar phosphate-isomerizing activities. Residue W97 functions as a molecular gate, opening and closing the intramolecular channel that connects the glutamine amide-hydrolysing and hexose phosphate-isomerizing domains
W97G
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almost complete elimination of the glucosamine 6-phosphate-synthetic activity, with the retention of the amidohydrolase and sugar phosphate-isomerizing activities. Residue W97 functions as a molecular gate, opening and closing the intramolecular channel that connects the glutamine amide-hydrolysing and hexose phosphate-isomerizing domains; mutation results in an almost complete elimination of the GlcN-6-P-synthetic activity, with the retention of the amidohydrolase and sugar phosphate-isomerizing activities
A38T
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mutant shows increased resistance against glucosamine-6-phosphate
A602L
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enhanced activity compared to the wild type enzyme, the behaviour of the mutant is similar to that of the wild type counterpart during purification demonstrating no significant modification in the overall protein structure
G471S
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mutant shows increased resistance against glucosamine-6-phosphate
I271T
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mutant shows increased resistance against glucosamine-6-phosphate
I3T
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mutant shows increased resistance against glucosamine-6-phosphate
L468P
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mutant shows increased resistance against glucosamine-6-phosphate
S449P
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mutant shows increased resistance against glucosamine-6-phosphate
V605L
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enhanced activity compared to the wild type enzyme, the behaviour of the mutant is similar to that of the wild type counterpart during purification demonstrating no significant modification in the overall protein structure
W74A
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efficiency of ammonia transfer is close to zero. No use of ammonia as a substrate; enhanced activity compared to the wild type enzyme, the behaviour of the mutant is similar to that of the wild type counterpart during purification demonstrating no significant modification in the overall protein structure
W74F
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enhanced activity compared to the wild type enzyme, the behaviour of the mutant is similar to that of the wild type counterpart during purification demonstrating no significant modification in the overall protein structure
W74L
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decrease in ammonia transfer, 5-7fold increase in the affinity for glutamine in the presence of fructose 6-phosphate; enhanced activity compared to the wild type enzyme, the behaviour of the mutant is similar to that of the wild type counterpart during purification demonstrating no significant modification in the overall protein structure
S243E
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increase in activity, 2fold lower Km value for D-fructose 6-phosphate than wild-type
biotechnology
the recombinant enzyme expressed in Saccharomyces cereviaise reveals some differences from the wild type enzyme, such as improved stability and less sensitivity to UDP-GlcNAc
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
medicine
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