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Information on EC 4.1.1.15 - glutamate decarboxylase and Organism(s) Escherichia coli and UniProt Accession P69908
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4.1.1.15 glutamate decarboxylaseIUBMB Comments
A pyridoxal-phosphate protein. The brain enzyme also acts on L-cysteate, 3-sulfino-L-alanine and L-aspartate.
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Word Map
- 4.1.1.15
-
gabaergic
-
gamma-aminobutyric
- autoimmune
- autoantibody
- islet
- mellitus
-
neurotransmitter
- synaptic
- hippocampus
-
interneurons
- cortical
- autoantigens
- choline
-
excitatory
-
dorsal
- striatum
-
insulin-dependent
-
ventral
- seizure
- parvalbumin
- epilepsy
- beta-cell
-
cholinergic
-
glutamatergic
- synapses
-
medial
-
neurotransmission
-
neurochemical
-
substantia
-
afferent
-
postsynaptic
- c-peptide
-
somata
-
chat
-
presynaptic
-
antibody-positive
-
autoreactive
- ketoacidosis
- bicuculline
- gaba-transaminase
- colliculus
-
preoptic
- gaba-t
-
calretinin
-
rostral
- muscimol
-
boutons
- reelin
- calbindin
- analysis
- synthesis
- pharmacology
- nutrition
- medicine
- food industry
- diagnostics
-
glycinergic
The taxonomic range for the selected organisms is: Escherichia coli
The expected taxonomic range for this enzyme is: Eukaryota, Bacteria, Archaea
The expected taxonomic range for this enzyme is: Eukaryota, Bacteria, Archaea
Synonyms
glutamic acid decarboxylase, gad65, gad67, glutamate decarboxylase, glutamic acid decarboxylase 65, glutamic acid decarboxylase 67, gad-65, gad-67, glutamate decarboxylase 67, l-glutamate decarboxylase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
65 kDa glutamic acid decarboxylase
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-
-
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67 kDa glutamic acid decarboxylase
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-
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Aspartate 1-decarboxylase
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-
-
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Aspartic alpha-decarboxylase
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-
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Cysteic acid decarboxylase
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-
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Decarboxylase, glutamate
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-
-
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ERT D1
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-
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GAD
GAD-65
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-
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GAD-67
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-
-
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GAD-alpha
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-
-
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GAD-beta
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-
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GAD-gamma
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-
-
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GadB
GADCase
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gamma-Glutamate decarboxylase
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GDCase
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Glutamic acid decarboxylase
Glutamic decarboxylase
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L-Aspartate-alpha-decarboxylase
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L-Glutamate alpha-decarboxylase
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L-Glutamate decarboxylase
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L-Glutamic acid decarboxylase
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L-Glutamic decarboxylase
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MGAD
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GAD
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Glutamic acid decarboxylase
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
decarboxylation
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-
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PATHWAY SOURCE
PATHWAYS
KEGG
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-, -, -
SYSTEMATIC NAME
IUBMB Comments
L-glutamate 1-carboxy-lyase (4-aminobutanoate-forming)
A pyridoxal-phosphate protein. The brain enzyme also acts on L-cysteate, 3-sulfino-L-alanine and L-aspartate.
CAS REGISTRY NUMBER
COMMENTARY
9024-58-2
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
additional information
?
-
-
GadB together with the antiporter gadC constitutes the gad acid resistance system, which confers the ability for bacterial survival for at least 2 h in a strongly acidic environment
-
-
?
L-glutamate
4-aminobutanoate + CO2
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-
-
-
?
L-glutamate
4-aminobutanoate + CO2
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the alpha-carboxyl group, leaving as CO2, is thus replaced by a cytoplasmic proton, yielding 4-aminobutanoate
-
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
additional information
?
-
-
GadB together with the antiporter gadC constitutes the gad acid resistance system, which confers the ability for bacterial survival for at least 2 h in a strongly acidic environment
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
pyridoxal 5'-phosphate
-
one molecule of pyridoxal 5'-phosphate is covalently bound to a lysyl residue
pyridoxal 5'-phosphate
-
the inactive mutant enzyme k276A contains very little pyridoxal 5'-phosphate, the inactive mutant enzyme K276H contains no pyridoxal 5'-phosphate
pyridoxal 5'-phosphate
PLP, plays a key role in the acquisition of a folding necessary for the canonical catalytic activity. Preparation of recombinant apoenzyme, and reconstitution with pyridoxal 5'-phosphate, overview
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Cl-
Mn2+
-
the optimal concentration (7.5 mM) of Mn2+ can also improve the activity of recombinant enzyme (164%), but the co-effect of Ca2+ and Mn2+ exhibits antagonism effect when added simultaneously
Cl-
binding of chloride ions to the N-terminus could stabilizes the triple-helix bundle
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
5,5'-dithiobis(2-nitrobenzoate)
-
0.1 mM, 50.3% inhibition at pH 4.6, irreversible
Chloroacetamide
-
no inhibition at pH 4.6, marked inhibition at pH 6.0 or higher
additional information
the C-terminal domain by entrancing into the active site is responsible for autoinhibition of the enzyme at neutral pH
-
additional information
-
the C-terminal domain by entrancing into the active site is responsible for autoinhibition of the enzyme at neutral pH
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
when commercial cation-exchange resins as solid acids, Amberlyst 15 and Amberlite IRC86, are simply added to the reaction medium, the conversion improves from 13% to 67% without salt formation. Even when water is used as the reaction medium, acidic ion-exchange resins enhance the reaction conversion significantly
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
additional information
-
cooperativeness is kept intact by residues Glu89 and His465 in the cooperativity system of GAD
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1.51
L-glutamate
mutant enzyme H465A, in 0.2 M pyridine/HCl buffer, pH 4.6, at 37°C
1.61
L-glutamate
mutant enzyme DELTAHT, in 0.2 M pyridine/HCl buffer, pH 4.6, at 37°C
2.32
L-glutamate
wild type enzyme, in 0.2 M pyridine/HCl buffer, pH 4.6, at 37°C
20.31
L-glutamate
pH 4.6, 37°C, recombinant C-terminally His-tagged wild-type enzyme
25.09
L-glutamate
pH 4.6, 37°C, recombinant deletion mutant GadBDELTA1-14
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
16.24
L-glutamate
mutant enzyme DELTAHT, in 0.2 M pyridine/HCl buffer, pH 4.6, at 37°C
20.75
L-glutamate
mutant enzyme H465A, in 0.2 M pyridine/HCl buffer, pH 4.6, at 37°C
24.85
L-glutamate
wild type enzyme, in 0.2 M pyridine/HCl buffer, pH 4.6, at 37°C
59.26
L-glutamate
pH 4.6, 37°C, recombinant C-terminally His-tagged wild-type enzyme
73.35
L-glutamate
pH 4.6, 37°C, recombinant deletion mutant GadBDELTA1-14
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
2.92
L-glutamate
pH 4.6, 37°C, recombinant C-terminally His-tagged wild-type enzyme and deletion mutant GadBDELTA1-14
10.08
L-glutamate
mutant enzyme DELTAHT, in 0.2 M pyridine/HCl buffer, pH 4.6, at 37°C
10.71
L-glutamate
wild type enzyme, in 0.2 M pyridine/HCl buffer, pH 4.6, at 37°C
13.74
L-glutamate
mutant enzyme H465A, in 0.2 M pyridine/HCl buffer, pH 4.6, at 37°C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
130.2
purified recombinant GAD obtained without supplementation (GAD-C), pH 4.8, 30°C
193.4
purified recombinant GAD produced in media supplemented with 0.05 mM soluble vitamin B6 analogue pyridoxine hydrochloride (GAD-V), pH 4.8, 30°C
additional information
additional information
-
4-aminobutanoate concentrations of 4.34-5.65 g/l in engineered Escherichia coli expressing enzymes GadA or GadB with antiporter GadC from different constructs, pH 6.2, 37°C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
3.8
4.6
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
3 - 7
purified recombinant enzyme, the activity decreases dramatically between pH 5.0 and pH 7.0. GAD-V exhibits a much higher activity than GAD-C below pH 5.0. More than 80% of the maximum catalytic activity is observed for GAD-V between pH 4.0 and pH 5.0, while GAD-C exhibits 98.3% and 38% of the activity shown by GAD-V at pH 5.0 and 4.0, respectively
3.5 - 5.5
GAD is active in the range pH 3.5 to pH 5.5 with optimal pH at 4.6, inactive above pH 6.0
additional information
pH rise caused by the reaction inactivates the enzyme catalyst, which is active only under acidic conditions, and consequently leads to low reaction conversions
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
30
37
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
-
localized exclusively in cytoplasm at neutral pH, but is recruited to the membrane when the pH falls
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localized exclusively in cytoplasm at neutral pH, but is recruited to the membrane when the pH falls
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
physiological function
in the GABA synthesis pathway GAD produces GABA from L-glutamate by promoting irreversible alpha-decarboxylation reaction as the most important and rate-limiting step
evolution
-
clustal X-generated dendrogram of bacterial glutamate decarboxylases, overview
malfunction
at pH values above pH 6.0, GAD is inactive due to conformational change of the hexameric enzyme at its N- and C-termini from acidic to neutral pH. Especially, His465 at the C-terminus of the enzyme together with Glu89 are demonstrated to be involved in the conformational change in a cooperative manner
physiological function
additional information
physiological function
-
the glutamate-dependent acid resistance (GDAR) system is by far the most potent acid resistance system in commensal and pathogenic Escherichia coli and requires the activity of intracellular glutamate decarboxylase GadB performing a proton-consuming decarboxylation reaction and the cognate antiporter GadC, which performs the glutamate/in/gamma-aminobutyrate/out electrogenic antiport, overview
physiological function
glutamate decarboxylase (GAD) catalyzes the irreversible decarboxylation of L-glutamate to the valuable food supplement gamma-aminobutyric acid (GABA)
additional information
molecular modelling of the active site, docking study, using the crystal structure of isoform A of Escherichia coli GAD (GADA) in complex with glutarate (as glutamate analogue) and pyridoxal 5'-phosphate, PDB ID 1XEY
additional information
-
molecular modelling of the active site, docking study, using the crystal structure of isoform A of Escherichia coli GAD (GADA) in complex with glutarate (as glutamate analogue) and pyridoxal 5'-phosphate, PDB ID 1XEY
additional information
-
at neutral pH the enzyme is in a compact conformation with access to the active site precluded by steric hindrance of some structural elements, a sequence of events lead to the conversion of GadB from the inactive into the active form and vice versa, structural determinants responsible for pH-dependent intracellular activation of GadB, overview. In its inactive form GadB has (i) the N-terminal residues 1-14 of each subunit mainly involved in dimerization and hexamerization, (ii) the C-terminal residues 452-466 ordered and protruding into the active site (with residues His465 and Thr466), like a plug, thus occupying the binding site of the physiological substrate glutamate, (iii) the beta-hairpin 300-313 contacting the C-terminal tail of the other subunit in the dimer as to hold it in place, structure comparisons, overview
additional information
the N-terminal fourteen residues (1-14) of homohexameric GadB forms a triple-helix bundle interdomain at acidic pH and contributes to the thermostability of GadB as the pH shifts from pH 7.6 to pH 4.6
additional information
-
the N-terminal fourteen residues (1-14) of homohexameric GadB forms a triple-helix bundle interdomain at acidic pH and contributes to the thermostability of GadB as the pH shifts from pH 7.6 to pH 4.6
additional information
pH rise caused by the reaction inactivates the enzyme catalyst, which is active only under acidic conditions, and consequently leads to low reaction conversions. Cross-linked aggregation method is used in order to extend the active range of GAD toward alkaline pH
additional information
three-dimensional enzyme structure analysis, homology modeling using the crystal structure of homohexameric GadB at low pH, PDB ID 1pmm
additional information
-
three-dimensional enzyme structure analysis, homology modeling using the crystal structure of homohexameric GadB at low pH, PDB ID 1pmm
PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dimer
hexamer
homohexamer
additional information
hexamer
-
GadB is a trimer of dimers, in which monomers from each dimer belong to different layers, structure comparisons, overview
additional information
Escherichia coli GAD forms a hexamer at acidic pH which consists of three functional dimers. In each dimer there are some special residues from both subunits that contribute in the formation of potential active sites and promote the interaction between the enzyme, cofactor and substrate. The N- and C-terminal domains of each subunit play an important role in conformational changes through pH shift. These conformational changes lead to activation of the enzyme at acidic pH and vice versa. The N-terminal residues involve in dimerization and subsequent migration of GAD to cytoplasmic site of the inner. The C-terminal domain by entrancing into the active site is also responsible for autoinhibition of the enzyme at neutral pH
additional information
-
Escherichia coli GAD forms a hexamer at acidic pH which consists of three functional dimers. In each dimer there are some special residues from both subunits that contribute in the formation of potential active sites and promote the interaction between the enzyme, cofactor and substrate. The N- and C-terminal domains of each subunit play an important role in conformational changes through pH shift. These conformational changes lead to activation of the enzyme at acidic pH and vice versa. The N-terminal residues involve in dimerization and subsequent migration of GAD to cytoplasmic site of the inner. The C-terminal domain by entrancing into the active site is also responsible for autoinhibition of the enzyme at neutral pH
additional information
homohexameric GadB forms a triple-helix bundle interdomain at acidic pH
additional information
-
homohexameric GadB forms a triple-helix bundle interdomain at acidic pH
additional information
addition of pyridoxine does not influence the aggregation state of GAD
additional information
in GadB, the dimer is the functional unit as each active site is made of amino acid residues that are provided by both monomers in the dimer. Structural organization of EcGadB in solution in the pH range 7.5-8.6, overview. Analysis by small angle X-ray scattering combined with size exclusion chromatography and analytical ultracentrifugation analysis shows that the compact and entangled EcGadB hexameric structure undergoes dissociation into dimers as pH alkalinizes. When pyridoxal 5'-phosphate is not present, the dimeric species is the most abundant in solution, though evidence for the occurrence of a likely tetrameric species is also obtained. Molecular modeling
additional information
-
in GadB, the dimer is the functional unit as each active site is made of amino acid residues that are provided by both monomers in the dimer. Structural organization of EcGadB in solution in the pH range 7.5-8.6, overview. Analysis by small angle X-ray scattering combined with size exclusion chromatography and analytical ultracentrifugation analysis shows that the compact and entangled EcGadB hexameric structure undergoes dissociation into dimers as pH alkalinizes. When pyridoxal 5'-phosphate is not present, the dimeric species is the most abundant in solution, though evidence for the occurrence of a likely tetrameric species is also obtained. Molecular modeling
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
enzyme hexamer X-ray diffraction structure determination and analysis at pH 4.6 and pH 7.6
-
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
E89A/H465A
-
site-directed mutagenesis, the double mutation not only brakes the cooperativity in the activity change but also yields a mutant enzyme that retains the activity at neutral pH. The resulting mutant enzyme, that is active at neutral pH, inhibits the cell growth in a glycerol medium by converting intracellular Glu into 4-aminobutanoate in an uncontrolled manner
E89Q
-
site-directed mutagenesis, double mutation Glu89Gln/DELTA452-466 strongly inhibits the cell growth and shows higher activity than mutant Glu89Gln/His465Ala
E89Q/H465A
-
site-directed mutagenesis, the double mutation not only brakes the cooperativity in the activity change but also yields a mutant enzyme that retains the activity at neutral pH. The resulting mutant enzyme, that is active at neutral pH, inhibits the cell growth in a glycerol medium by converting intracellular Glu into 4-aminobutanoate in an uncontrolled manner
H465A
K276A
-
no decarboxylation of L-Glu. Transition temperature is 11°C higher than that of the wild-type enzyme. Limited proteolysis by trypsin shows that the mutant enzyme is more resistant to proteolytic degradation than the wild-type enzyme. Mutant enzyme contains very little pyridoxal 5'-phosphate
K276H
-
no decarboxylation of L-Glu. Transition temperature is 4°C higher than that of the wild-type enzyme. Mutant enzyme contains no pyridoxal 5'-phosphate
Q5D/V6I/T7E
site-directed mutagenesis the mutant shows higher thermostability and increased melting temperature compared to the wild-type, but shows no reduction of catalytic activity
Q5I/V6D/T7Q
mutant M6, site-directed mutagenesis, the mutant shows higher thermostability, with a 5.6times (560%) increase in half-life value at 45°C, 8.7°C rise in melting temperature (Tm) and a 14.3°C rise in the temperature at which 50% of the initial activity remained after 15 min incubation (T15/50), compared to wild-type enzyme. The induced new hydrogen bonds in the same polypeptide chain or between polypeptide chains in Escherichia coli GadB homohexamer may be responsible for the improved thermostability. Increased thermostability contributes to increased GABA conversion ability. After 12 h conversion of 3 mol/l glutamate, 297 g/l GABA is produced and 95% mole conversion rate is catalyzed by mtant M6 whole cells while those by wild-type GAD are 273.5 g/L and 86.2%, respectively
Q5N/V6Y/T7V
mutant M1, site-directed mutagenesis, the mutant shows improved thermostability and increased activity compared to the wild-type enzyme
Q5Y/V6R/T7K
mutant M8, site-directed mutagenesis, the mutant shows improved thermostability and increased activity compared to the wild-type enzyme
additional information
H465A
the mutant is active at pH well above 5.7 and shows 78% of wild type specific activity in 0.2 M pyridine/HCl buffer, pH 4.6, at 37°C in the presence of 0.1 mM pyridoxal 5'-phosphate
additional information
point mutation is performed virtually in the active site of the Escherichia coli GAD in order to increase thermal stability and catalytic activity of the enzyme, overview. Molecular modelling results indicate that performing mutation separately at positions 164, 302, 304, 393, 396, 398 and 410 increase binding affinity to substrate. The enzyme is predicted to be more thermostable in all 7 mutants based on DDG value. Stabilizing mutations in the active site based on DDG value, and binding energy levels, overview. Cavity volume change analysis for selected mutants
additional information
-
point mutation is performed virtually in the active site of the Escherichia coli GAD in order to increase thermal stability and catalytic activity of the enzyme, overview. Molecular modelling results indicate that performing mutation separately at positions 164, 302, 304, 393, 396, 398 and 410 increase binding affinity to substrate. The enzyme is predicted to be more thermostable in all 7 mutants based on DDG value. Stabilizing mutations in the active site based on DDG value, and binding energy levels, overview. Cavity volume change analysis for selected mutants
additional information
the mutant GadBDELTAHT (His465 of GadB is deleted together with the last residue in the polypeptide chain, Thr466) is active at pH well above 5.7 and shows 52% of wild type specific activity in 0.2 M pyridine/HCl buffer, pH 4.6, at 37°C in the presence of 0.1 mM pyridoxal 5'-phosphate
additional information
-
the mutant GadBDELTAHT (His465 of GadB is deleted together with the last residue in the polypeptide chain, Thr466) is active at pH well above 5.7 and shows 52% of wild type specific activity in 0.2 M pyridine/HCl buffer, pH 4.6, at 37°C in the presence of 0.1 mM pyridoxal 5'-phosphate
additional information
-
construction of a synthetic protein complex to improve the 4-aminobutanoate conversion in engineered Escherichia coli strain XL1-Blue, by assembling a single protein-protein interaction domain SH3 to the glutamate decarboxylase (GadA and GadB) and attaching a cognate peptide ligand to the glutamate/4-aminobutanoate antiporter (GadC) at the N-terminus, C-terminus, and the 233rd amino acid residue
additional information
-
expanding the active pH range of the enzyme by breaking the cooperativeness
additional information
-
generation of a DELTA452-466 deletion mutant, the mutant can convert glycerol into 4-aminobutanoate with minimal growth inhibition to maximize its space-time yield. Double mutation Glu89Gln/DELTA452-466 strongly inhibits the cell growth and shows higher activity than mutant Glu89Gln/His465Ala
additional information
-
immobilization of the cellulose binding domain-enzyme fusion protein on a crystalline cellulose resin, with binding capacity of 33 nmol CBD-GAD/g resin, the immobilized enzymes retains 60% of initial activiy after 10 uses
additional information
improvement of the thermostability of GadB through structural optimization ofits N-terminal interdomain. Residues Gln5, Val6, and Thr7 are potential mutational target sites for the optimization of inter- and intra-molecular interactions of the triple-helix bundle. Generation of a deletion mutant of GadB lacking residues 1-14 at the N-terminus, GadBDELTA1-14
additional information
-
improvement of the thermostability of GadB through structural optimization ofits N-terminal interdomain. Residues Gln5, Val6, and Thr7 are potential mutational target sites for the optimization of inter- and intra-molecular interactions of the triple-helix bundle. Generation of a deletion mutant of GadB lacking residues 1-14 at the N-terminus, GadBDELTA1-14
additional information
application of site-directed saturation mutagenesis of the N-terminal residues of GadB from Escherichia coli to improve its thermostability. Among the mutants tested, M6 is the most thermostable one
additional information
-
application of site-directed saturation mutagenesis of the N-terminal residues of GadB from Escherichia coli to improve its thermostability. Among the mutants tested, M6 is the most thermostable one
additional information
-
Corynebacterium glutamicum, the major L-glutamate producing microorganism, has been engineered to achieve direct fermentative production of GABA from glucose, method optimization, overview. In the fed-batch cultivation at pH 5.0, which is not preferable condition for cell growth, much less glutamate might be synthesized, which results in much lower production of GABA compared with that of pH 6.0. In the culture at pH 6.0, increased GAD activity can be balanced for glutamate synthesis to result in high-level production of GABA of up to 38.6 g/l. Corynebacterium glutamicum expressing Escherichia coli GAD mutant under the strong PH36 promoter produces GABA to the concentration of 5.89 g/l in GP1 medium at pH 7.0, which is 17fold higher than that obtained by Corynebacterium glutamicum expressing wild-type Escherichia coli GAD in the same condition (0.34 g/l). Optimized fed-batch culture of Corynebacterium glutamicum expressing Escherichia coli GAD mutant in GP1 medium containing 0.05 mg/l of biotin at pH 6.0, 30°C, results in the highest GABA concentration of 38.6 g/l with the productivity of 0.536 g/l/h
additional information
cross-linked aggregation method is used in order to extend the active range of GAD toward alkaline pH. Cross-linked aggregation activate GAD even at neutral and alkaline pH values. It is a useful method capable of facilitating recovery and reuse of the enzyme as well as increasing the reaction conversion by extending the active pH range of GAD. GAD from Escherichia coli is prepared as cross-linked enzyme aggregate (CLEA) in which the enzyme is precipitated using ammonium sulfate (60% saturation) and then cross-linked with glutaraldehyde (2%) in sodium acetate buffer (0.2 mol/l, pH 4.6). The cross-linked aggregation extends an active pH-range of GAD from pH 5.5 up to pH 8.0. As a result, the reaction conversion of 1 mol/l monosodium L-glutamate into GABA is improved from 13% to 22%. Moreover, the CLEA of GAD is easily recovered after the reaction and reused retaining over 95% of its initial activity during the first four cycles and over 60% activity at the 10th cycle. Method evaluation and optimization, overview
additional information
enhanced production of recombinant Escherichia coli glutamate decarboxylase through optimization of induction strategy and addition of pyridoxine, different induction strategies are investigated, induction is optimal when the temperature is maintained at 30°C, the inducer lactose is fed at a rate of 0.2 g/l/h, and protein expression is induced when the cell density (OD600) reaches 50. Under these conditions, the GAD activity of 1273.8 U/ml is achieved. The supplementing the medium with 2 mM pyridoxine hydrochloride (PN), a cheap and stable PLP precursor, at the initiation of protein expression, and then again 10 h later, results in very high GAD activity of 3193.4 U/ml. Fed-batch cultivation in a 3.6-L fermentor at 37°C and pH 7.0
additional information
-
enhanced production of recombinant Escherichia coli glutamate decarboxylase through optimization of induction strategy and addition of pyridoxine, different induction strategies are investigated, induction is optimal when the temperature is maintained at 30°C, the inducer lactose is fed at a rate of 0.2 g/l/h, and protein expression is induced when the cell density (OD600) reaches 50. Under these conditions, the GAD activity of 1273.8 U/ml is achieved. The supplementing the medium with 2 mM pyridoxine hydrochloride (PN), a cheap and stable PLP precursor, at the initiation of protein expression, and then again 10 h later, results in very high GAD activity of 3193.4 U/ml. Fed-batch cultivation in a 3.6-L fermentor at 37°C and pH 7.0
additional information
expression of gadB2 produced more GABA in Corynebacterium glutamicum than expression of gadB1. Construction of gadB2 expression strains under different RBS sequence and promoters (tacM, sglB, hmp, ilvE, cg1417, gapA, dtsR, tuf, cspB, odhL, uspA, gdh, sod, and pqo), engineering of RBS sequence and promoter, evaluation of GABA production, overview. In recombinant Corynebacterium glutamicum, the optimal pH for cell growth and Glu biosynthesis is about 7.0, whereas that for GAD activity and conversion of Glu to GABA is 5.0-6.0
additional information
expression of gadB2 produced more GABA in Corynebacterium glutamicum than expression of gadB1. Construction of gadB2 expression strains under different RBS sequence and promoters (tacM, sglB, hmp, ilvE, cg1417, gapA, dtsR, tuf, cspB, odhL, uspA, gdh, sod, and pqo), engineering of RBS sequence and promoter, evaluation of GABA production, overview. In recombinant Corynebacterium glutamicum, the optimal pH for cell growth and Glu biosynthesis is about 7.0, whereas that for GAD activity and conversion of Glu to GABA is 5.0-6.0
additional information
GABA is produced from glutamate through decarboxylation catalyzed by the recombinant glutamate decarboxylase (GAD) expressed in Escherichia coli strain BL21(DE3), the GAD-catalyzed reaction is conducted in 0.2 mol/l sodium acetate buffer (pH 4.6) with 1 mol/l monosodium glutamate at 37°C, optimization of GABA production method, overview. When commercial cation-exchange resins as solid acids are simply added to the reaction medium, the conversion improves from 13% to 67% without salt formation. Even when water is used as the reaction medium, acidic ion-exchange resins enhance the reaction conversion significantly. In a salt-free manner, acidic resins suppress the pH rise during the reaction so that they can enhance the reaction conversion. In addition, they can be recovered and reused easily after the reaction. Heterogeneous solid acids make the GABA production processmore economical and eco-friendly
additional information
optimization of the reaction conditions for recombinant GABA production by the homogenously expressed enzyme from Escherichia coli. The activity for GAD produced in media supplemented with 0.05 mM soluble vitamin B6 analogue pyridoxine hydrochloride (GAD-V) is 154.8 U/l, 1.8fold higher than that of GAD obtained without supplementation (GAD-C). Purified GAD-V exhibits increased activity of 193.4 U/mg (1.5fold higher), superior thermostability (2.8fold greater), and higher kcat/Km (1.6fold higher) compared to GAD-C. Under optimal conditions in reactions mixtures lacking added pyridoxal 5'-phosphate, crude GAD-V converts 500 g/l monosodium glutamate to GABA with a yield of 100%, and 750 g/l monosodium glutamate with a yield of 88.7%. Effect of substrate concentration on GABA production by GAD-C and GAD-V, overview
additional information
production method optimization, overexpression of Escherichia coli gene gadB in strain BL21(DE3), overview. Some GadB proteins overexpressed in BL21(DE3)/pET20b-pelB-gadB might not be functional. The highest extracellular GadB activity (1.39 U/mL) is found in BL21(DE3)/pET20b-torAgadB with 0.7 mM IPTG induction
additional information
-
production method optimization, overexpression of Escherichia coli gene gadB in strain BL21(DE3), overview. Some GadB proteins overexpressed in BL21(DE3)/pET20b-pelB-gadB might not be functional. The highest extracellular GadB activity (1.39 U/mL) is found in BL21(DE3)/pET20b-torAgadB with 0.7 mM IPTG induction
additional information
the Escherichia coli glutamate decarboxylase is expressed in Corynebacterium glutamicum for production of gamma-aminobutyric acid (GABA), a building block of the biodegradable plastic polyamide 4. Disruption of gene pknG, encoding serine/threonine protein kinase G (EC 2.7.11.1), enhances recombinant production of gamma-aminobutyric acid through reduction of the 2-oxoglutarate dehydrogenase complex (ODHC) activity regulation, PknG catalyzes the phosphorylation of OdhI, a 15 kDa subunit of ODHC, which reverses the inhibition of the ODHC activity. Strain GAD produces 13.06 g/l of GABA in 120 hours, consuming 83.62 g/l of glucose, strain GADDELTApknG produces 0.272 g/l of GABA in 120 hours, consuming less glucose
additional information
-
the Escherichia coli glutamate decarboxylase is expressed in Corynebacterium glutamicum for production of gamma-aminobutyric acid (GABA), a building block of the biodegradable plastic polyamide 4. Disruption of gene pknG, encoding serine/threonine protein kinase G (EC 2.7.11.1), enhances recombinant production of gamma-aminobutyric acid through reduction of the 2-oxoglutarate dehydrogenase complex (ODHC) activity regulation, PknG catalyzes the phosphorylation of OdhI, a 15 kDa subunit of ODHC, which reverses the inhibition of the ODHC activity. Strain GAD produces 13.06 g/l of GABA in 120 hours, consuming 83.62 g/l of glucose, strain GADDELTApknG produces 0.272 g/l of GABA in 120 hours, consuming less glucose
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
3 - 7
recombinant enzyme, 4°C, 24 h, both GAD-C and GAD-V retain more than 70% of their maximal activity at pH 3.0-5.0 and more than 80% at pH 5.0-7.0
749087
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
100
37
recombinant enzyme in Escherichia coli cells, pH 4.6, loss of 50% activity after 3 h, of 70% activity after 12 h, and of 80% activity after 36 h
40
after 12 h incubation at 40°C, the residual activity of mutants M1, M6 and M8 is 52.05%, 65.08% and 30.2%, respectively, about 4.16, 5.2 and 2.4fold that of wild-type GadB (12.5%)
45
half-life of recombinant wild-type GadB is 24.24 min, while the half-lives of mutants M1, M6 and M8 are 128.84 min, 160.45 min and 126.03 min, respectively. The half-lives of M1, M6 and M8 are 5.32, 6.62 and 5.20fold that of the wild-type enzyme
50
50 - 60
after 12 h incubation at 50°C and 60°C, the residual activity of wild-type GadB is 12.5% and 9.0%, respectively. The residual activity of mutants M1, M6 and M8 is 35.16% and 27.17%, 38.10% and 33.25%, 27.19% and 24.39%, respectively, which is at least 2fold higher than that of wild-type GadB
55
additional information
50
recombinant enzyme in Escherichia coli cells, pH 4.6, loss of about 70% activity after 3 h, of over 80% activity after 12 h, and of over 90% activity after 36 h
55
purified recombinant wild-type enzyme, without chloride ions, 10 min, 71% activity remaining at pH 4.6, 2% remaining at pH 8.0, the deletion mutant GadB DELTA1-14 exhibits similar thermostabilityat pH 4.6 and pH 8.0
additional information
homohexameric GadB forms a triple-helix bundle interdomain at acidic pH and contributes to the thermostability of GadB
additional information
-
homohexameric GadB forms a triple-helix bundle interdomain at acidic pH and contributes to the thermostability of GadB
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
by entrapping Escherichia coli glutamate decarboxylase into sodium alginate and carrageenan gel beads, the activity of immobilized GAD remains 85% during the initial five batches and the activity still remains 50% at the tenth batch
-
limited proteolysis by trypsin shows that the mutant enzyme is more resistant to proteolytic degradation than the wild-type enzyme
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
in polyethylene glycol (PEG) and sodium sulfate aqueous two-phase system. The optimum system obtained for GAD purification is composed of PEG 4000, tie line length of 63.5%, a volume ratio of 2.31, a loading sample concentration of 0.4 g/ml, which produces a GAD recovery of 90% with the purification fold of 73. The purification fold declines from 71.73 to 23.27 with the addition of 1 mM NaCl
-
purification of recombinant holoenzyme, preparation of apoenzyme, and reconstitution with pyridoxal 5'-phosphate, overview
recombinant C-terminally His-tagged wild-type and mutant enzyme from Escherichia coli strain BL21(DE3) by affinity chromatography and ultrafiltration
recombinant enzyme 1.7-1.9fold from Escherichia coli strain BL21(DE3) by ammonium sulfate fractionation, dialysis, anion exchange chromatographyand again dialysis, to homogeneity
recombinant enzyme from Escherichia coli strain BL21(DE3) partially by ammonium sulfate fractionation
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and desalting gel filtration, to over 90% purity
recombinant His6-tagged fusion enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
gene gabB, recombinant expression in Escherichia coli strain BL21(DE3), intracellular and extracellular expression of GadB from BL21(DE3)/pET20b-gadB, BL21(DE3)/pET20b-pelBgadB, and BL21(DE3)/pET20b-torA-gadB, subcloning in Escherichia coli strain JM109
gene gad, cloning in Escherichia coli strain DH5alpha, overexpression of His-tagged wild-type and mutant in Escherichia coli strain BL21(DE3)
-
gene gad, recombinant lactose-induced overexpression of enzyme GAD in Escherichia coli strain BL21(DE3)
gene gad, the enzyme is fused to the cellulose-binding domain and a linker of Trichoderma harzianum endoglucanase II, or S3N10 peptide replacing the native linker to prevent proteolytic cleavage, functional expression of the His6-tagged fusion construct in Escherichia coli strain BL21(DE3)
-
gene gadB, expression of C-terminally His-tagged wild-type and mutant enzyme in Escherichia coli strain BL21(DE3), cloning in Escherichia coli strain DH5alpha
gene gadB, recombinant cytoplasmic expression in Escherichia coli strain BL21(DE3), using vectors pMD18-T and pET-24a(+) and IPTG induction, subcloning in Escherichia coli strain JM109
gene gadB, recombinant expression in Corynebacterium glutamicum strain ATCC 13032 with or without gene pknG knockout
gene gadB, recombinant expression in Escherichia coli strain BL21(DE3)
gene gadB, recombinant expression in Escherichia coli strain BL21(DE3), subcloning in Escherichia coli strain DH5alpha
gene gadB, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3), subcloning in Escherichia coli strain DH5alpha
gene gadB, recombinnat enzyme expression in Corynebacterium glutamicum, evaluation of PH36, PI16, and PL26 promoters for optimal expression and different pH value for engineered cultures
-
gene gadB1, recombinant expression in Corynebacterium glutamicum strain SH, subcloning in Escherichia coli strain JM109
gene gadB2, recombinant expression in Corynebacterium glutamicum strain SH, subcloning in Escherichia coli strain JM109. Construction of gadB2 expression strains under different RBS sequence and promoter, expression of gadB2 with bicistronic expression cassette, overview
genes gadA and gadB, co-overexpression of the enzyme fused to a protein-protein interaction domain SH3 in Escherichia coli strain XL1-Blue with gene gadC encoding an antiporter
-
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
expression of the gadBC operon increases under conditions of respiratory stress
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
medicine
-
incubation of rat hippocampal slices with the potassium channel antagonis tetraethyl ammonium results in widespread excitotoxic death of pyramidal and granule cell neurons. Treatment with bacterial enzyme significantly reduces excitotoxicity induced by tetraethyl ammonium without showing neurotoxicity. Targeting of enzyme to the interior of synaptic vesicles may enhance its potency as a neuroprotectant
synthesis
-
the enzyme is a key component of 4-aminobutanoate production through an enzymatic process
REF.
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TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
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L-Glutamate decarboxylase from bacteria
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1985
Clostridium perfringens, Escherichia coli
Fields, H.A.; Wheeler, C.M.; Davis, C.L.; Warner, T.N.; Bradley, D.W.; Maynard, J.E.
Purification and radiometric assay detection of glutamate decarboxylase
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1982
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Youngs, T.L.; Tunnicliff, G.
Substrate analogues and divalent cations as inhibitors of glutamate decarboxylase from Escherichia coli
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Escherichia coli, Mus musculus
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Ornithine and glutamate decarboxylases catalyze an oxidative deamination of their a-methyl substrates
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Escherichia coli
-
McCormick, S.J.; Tunnicliff, G.
Kinetics of inactivation of glutamate decarboxylase by cysteine-specific reagents
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Escherichia coli
Capitani, G.; De Biase, D.; Aurizi, C.; Gut, H.; Bossa, F.; Grutter, M.G.
Crystal structure and functional analysis of Escherichia coli glutamate decarboxylase
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22
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2003
Escherichia coli
Drsata, J.; Netopilova, M.; Tolman, V.
Stereoisomers of 4-fluoroglutamic acid. Influence on Escherichia coli glutamate decarboxylase
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1999
Escherichia coli
Dutyshev, D.I.; Darii, E.L.; Fomenkova, N.P.; Pechik, I.V.; Polyakov, K.M.; Nikonov, S.V.; Andreeva, N.S.; Sukhareva, B.S.
Structure of Escherichia coli glutamate decarboxylase (GADalpha) in complex with glutarate at 2.05 A resolution
Acta Crystallogr. Sect. D
61
230-235
2005
Escherichia coli
Matthews, C.C.; Zielke, H.R.; Fishman, P.S.; Remington, M.P.; Bowen, T.G.
Glutamate decarboxylase protects neurons against excitotoxic injury
J. Neurosci. Res.
85
855-859
2007
Escherichia coli
Pennacchietti, E.; Lammens, T.M.; Capitani, G.; Franssen, M.C.; John, R.A.; Bossa, F.; De Biase, D.
Mutation of His465 alters the pH-dependent spectroscopic properties of Escherichia coli glutamate decarboxylase and broadens the range of its activity toward more alkaline pH
J. Biol. Chem.
284
31587-31596
2009
Escherichia coli (P69910), Escherichia coli
Yao, W.; Wu, X.; Zhu, J.; Sun, B.; Miller, C.
System establishment of ATPS for one-step purification of glutamate decarboxylase from E. coli after cell disruption
Appl. Biochem. Biotechnol.
164
1339-1349
2011
Escherichia coli
Wang, Q.; Xin, Y.; Zhang, F.; Feng, Z.; Fu, J.; Luo, L.; Yin, Z.
Enhanced ?-aminobutyric acid-forming activity of recombinant glutamate decarboxylase (gadA) from Escherichia coli
World J. Microbiol. Biotechnol.
27
693-700
2011
Escherichia coli
Park, H.; Ahn, J.; Lee, J.; Lee, H.; Kim, C.; Jung, J.K.; Lee, H.; Lee, E.G.
Expression, immobilization and enzymatic properties of glutamate decarboxylase fused to a cellulose-binding domain
Int. J. Mol. Sci.
13
358-368
2012
Escherichia coli
Thu Ho, N.A.; Hou, C.Y.; Kim, W.H.; Kang, T.J.
Expanding the active pH range of Escherichia coli glutamate decarboxylase by breaking the cooperativeness
J. Biosci. Bioeng.
115
154-158
2013
Escherichia coli
Jun, C.; Joo, J.C.; Lee, J.H.; Kim, Y.H.
Thermostabilization of glutamate decarboxylase B from Escherichia coli by structure-guided design of its pH-responsive N-terminal interdomain
J. Biotechnol.
174
22-28
2014
Escherichia coli (P69910), Escherichia coli
Le Vo, T.D.; Ko, J.S.; Park, S.J.; Lee, S.H.; Hong, S.H.
Efficient gamma-aminobutyric acid bioconversion by employing synthetic complex between glutamate decarboxylase and glutamate/GABA antiporter in engineered Escherichia coli
J. Ind. Microbiol. Biotechnol.
40
927-933
2013
Escherichia coli
De Biase, D.; Pennacchietti, E.
Glutamate decarboxylase-dependent acid resistance in orally acquired bacteria: function, distribution and biomedical implications of the gadBC operon
Mol. Microbiol.
86
770-786
2012
Escherichia coli, Listeria monocytogenes, Levilactobacillus brevis (A9ZM78), Levilactobacillus brevis (Q03U69), Levilactobacillus brevis ATCC 367 (Q03U69), Levilactobacillus brevis FO12005 (A9ZM78)
Okai, N.; Takahashi, C.; Hatada, K.; Ogino, C.; Kondo, A.
Disruption of pknG enhances production of gamma-aminobutyric acid by Corynebacterium glutamicum expressing glutamate decarboxylase
AMB Express
4
20
2014
Escherichia coli (A5YKJ2), Escherichia coli
Zhao, A.; Hu, X.; Li, Y.; Chen, C.; Wang, X.
Extracellular expression of glutamate decarboxylase B in Escherichia coli to improve gamma-aminobutyric acid production
AMB Express
6
55
2016
Escherichia coli (P69910), Escherichia coli, Escherichia coli K-12 / W3110 (P69910)
Shi, F.; Luan, M.; Li, Y.
Ribosomal binding site sequences and promoters for expressing glutamate decarboxylase and producing gamma-aminobutyrate in Corynebacterium glutamicum
AMB Express
8
61
2018
Escherichia coli (A0A0J2E2M6), Escherichia coli (A0A2Y0HLE8)
Su, L.; Huang, Y.; Wu, J.
Enhanced production of recombinant Escherichia coli glutamate decarboxylase through optimization of induction strategy and addition of pyridoxine
Biores. Technol.
198
63-69
2015
Escherichia coli (P69910), Escherichia coli
Tavakoli, Y.; Esmaeili, A.; Saber, H.
Increasing thermal stability and catalytic activity of glutamate decarboxylase in E. coli an in silico study
Comput. Biol. Chem.
64
74-81
2016
Escherichia coli (P69908), Escherichia coli
Fan, L.Q.; Li, M.W.; Qiu, Y.J.; Chen, Q.M.; Jiang, S.J.; Shang, Y.J.; Zhao, L.M.
Increasing thermal stability of glutamate decarboxylase from Escherichia coli by site-directed saturation mutagenesis and its application in GABA production
J. Biotechnol.
278
1-9
2018
Escherichia coli (A5YKJ2), Escherichia coli
Dinh, T.; Ho, N.; Kang, T.; Mcdonald, K.; Won, K.
Salt-free production of gamma-aminobutyric acid from glutamate using glutamate decarboxylase separated from Escherichia coli
J. Chem. Technol. Biotechnol.
89
1432-1436
2014
Escherichia coli (P69910)
-
Dinh, T.; Jang, N.; Mcdonald, K.; Won, K.
Cross-linked aggregation of glutamate decarboxylase to extend its activity range toward alkaline pH
J. Chem. Technol. Biotechnol.
90
2100-2105
2015
Escherichia coli (P69910)
-
Choi, J.W.; Yim, S.S.; Lee, S.H.; Kang, T.J.; Park, S.J.; Jeong, K.J.
Enhanced production of gamma-aminobutyrate (GABA) in recombinant Corynebacterium glutamicum by expressing glutamate decarboxylase active in expanded pH range
Microb. Cell Fact.
14
21
2015
Escherichia coli
Huang, Y.; Su, L.; Wu, J.
Pyridoxine supplementation improves the activity of recombinant glutamate decarboxylase and the enzymatic production of gama-aminobutyric acid
PLoS ONE
11
e0157466
2016
Escherichia coli (P69910), Escherichia coli K-12 / MG1655 (P69910)
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On the effect of alkaline pH and cofactor availability in the conformational and oligomeric state of Escherichia coli glutamate decarboxylase
Protein Eng. Des. Sel.
30
235-244
2017
Escherichia coli (P69910), Escherichia coli
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