Information on EC 1.4.1.14 - glutamate synthase (NADH)

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

EC NUMBER
COMMENTARY hide
1.4.1.14
-
RECOMMENDED NAME
GeneOntology No.
glutamate synthase (NADH)
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
2 L-glutamate + NAD+ = L-glutamine + 2-oxoglutarate + NADH + H+
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
-
-
-
-
redox reaction
-
-
-
-
reduction
-
-
-
-
transamidation
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
ammonia assimilation cycle I
-
-
L-glutamate biosynthesis IV
-
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glutamate and glutamine metabolism
-
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Alanine, aspartate and glutamate metabolism
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Nitrogen metabolism
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Metabolic pathways
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Biosynthesis of secondary metabolites
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Microbial metabolism in diverse environments
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Biosynthesis of antibiotics
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SYSTEMATIC NAME
IUBMB Comments
L-glutamate:NAD+ oxidoreductase (transaminating)
A flavoprotein (FMN).
CAS REGISTRY NUMBER
COMMENTARY hide
65589-88-0
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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-
-
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
two isozymes encoded by genes gltA and gltA2 of the gltAB operon
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-
Manually annotated by BRENDA team
two isozymes encoded by genes gltA and gltA2 of the gltAB operon
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-
Manually annotated by BRENDA team
strain CCAP 11/32a
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-
Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
strain ATCC 6013
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-
Manually annotated by BRENDA team
obligate aerobic bacterium
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-
Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
Z
-
-
Manually annotated by BRENDA team
Z
-
-
Manually annotated by BRENDA team
strain am-1
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-
Manually annotated by BRENDA team
strain X2180A
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-
Manually annotated by BRENDA team
serovar typhimurium
-
-
Manually annotated by BRENDA team
Sf9
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-
Manually annotated by BRENDA team
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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
2 L-glutamate + NAD+
L-glutamine + 2-oxoglutarate + NADH + H+
show the reaction diagram
L-glutamine + 2-oxoglutarate + NADH
glutamate + NAD+
show the reaction diagram
L-glutamine + 2-oxoglutarate + NADH
L-glutamate + NAD+
show the reaction diagram
L-glutamine + 2-oxoglutarate + reduced form of 3-acetylpyridine adenine dinucleotide
L-glutamine + oxidized form of 3-acetylpyridine adenine dinucleotide
show the reaction diagram
-
reduced form of 3-acetylpyridine adenine dinucleotide is an alternative reductant to NADH, binds as tightly as NADH, enzyme catalyzes NADH-dependent reduction of AcPdAD+ by a substituted-enzyme ping-pong mechanism
-
?
additional information
?
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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
2 L-glutamate + NAD+
L-glutamine + 2-oxoglutarate + NADH + H+
show the reaction diagram
L-glutamine + 2-oxoglutarate + NADH
glutamate + NAD+
show the reaction diagram
additional information
?
-
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enzyme is important for nitrogen assimilation, reduced enzyme ativity impairs N assimilation, overview
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-
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
3-acetylpyridine adenine dinucleotide
-
reduced form is an alternative reductant, binds as tightly as NADH
flavin
additional information
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3Fe-4S-center
Cd2+
-
0.5 mM increases GOGAT activity in the presence of 2-oxoglutarate
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(2,5-dioxoimidazolidin-4-yl)urea
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activity of NADH-GOGAT I significantly decreases in the presence of 10 mM allantoine
(2S)-2-amino-4-(S-methylsulfonimidoyl)butanoic acid
2-Hydroxyglutarate
-
D- and L-isomer, most potent competitive inhibitor
2-oxoglutarate
-
-
3-acetylpyridine adenine dinucleotide
-
competitive inhibitor with respect to NADH
6-diazo-5-oxo-L-norleucine
acridine
-
inhibitor of flavoenzymes, 65% inhibition
allopurinol
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activity of NADH-GOGAT I significantly decreases in the presence of 2 mM allopurinol
arginine
-
20 mM, complete inhibition
asparagine
aspartate
azaserine
bathophenanthroline
-
50% inhibition
Cd2+
-
0.5 mM slightly decreases GOGAT activity in the absence of 2-oxoglutarate
citrate
-
inhibited by high concentrations, 40 mM
Co2+
-
slight inhibition
Cu2+
-
Cu2+ and Fe2+ are most inhibitory metal ions
D-2-hydroxyglutarate
-
-
D-glutamate
-
competitive inhibitor
D-glutamine
DL-ethionine sulfone
DL-homocysteic acid
-
-
Fe2+
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Cu2+ and Fe2+ are most inhibitory metal ions
glutamine
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activity of NADH-GOGAT I significantly decreases in the presence of 10 mM glutamine
Glutarate
-
most potent competitive inhibitor
glycine
L-2-Amino-4-oxo-5-chloropentanoate
-
1 mM, 30 min, 0C, pH 7.6, irreversible inhibition
L-2-Hydroxyglutarate
-
-
L-Albizziin
L-glutamate
L-histidine
-
25 mM, 20-30% inhibition
L-homocysteine sulfonamide
-
very potent transition state inhibitor, competitive to L-glutamine
L-methionine D,L-sulfoximine
-
-
L-Methionine sulfone
L-methionine-SR-sulfoxide
leucine
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10 mM, 52% inhibition
malate
methionine
Mg2+
-
slight inhibition
NADH
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substrate inhibition at high concentrations
NADPH
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0.2 mM, 50% inhibition
O-diazoacetyl-L-serine
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1 mM, 30 min, 0C, pH 7.6, irreversible inhibition
oxaloacetate
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weak inhibition, competitive with 2-oxoglutarate
p-hydroxymercuribenzoate
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complete inhibition at 0.1-1 mM, at 0.0005 mM 33% inhibition or 75% inhibition after preincubation in presence of glutamine and 2-oxoglutarate, substrates induce a conformation, which makes essential sulfhydryl groups more accessible to reagent
phenylalanine
-
20 mM, complete inhibition
pyruvate
serine
Sodium dithionite
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strong inhibitory effect
tryptophan
xanthine
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activity of NADH-GOGAT I significantly decreases in the presence of 10 mM xanthine
Zn2+
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slight inhibition
additional information
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-mercaptoethanol
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1%, presence of a reducing agent is essential for enzyme activity
diphosphate
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5 mM, stimulates activity with 36% enhancement
dithiothreitol
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0.5 mg per ml, presence of a reducing agent is essential for enzyme activity
GltB
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GltA and not GltA2 is dependent on GltB for GOGAT activity in vivo
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NaNO3
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transcripts for NADH-GOGAT transiently accumulate when 1 mM is applied
NH4Cl
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NADH-GOGAT gene expression is transiently induced within a few hours of supplying either 1 or 25 mM to the medium, leading to a marked accumulation of NADH-GOGAT mRNA
NH4NO3
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transcripts for NADH-GOGAT transiently accumulate when 0.5 mM is applied
succinate
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50 mM, stimulates activity by 57%
additional information
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0014 - 0.27
2-oxoglutarate
0.003 - 0.014
3-acetylpyridine adenine dinucleotide
0.001
L-glutamate
-
pH 8.5, 45C, GltA and GltA2
0.125 - 1
L-glutamine
0.00124 - 0.11
NADH
additional information
additional information
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-
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
18 - 51
3-acetylpyridine adenine dinucleotide
32 - 70
L-glutamine
70
NADH
Lupinus angustifolius
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-
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.146
(2S)-2-amino-4-(S-methylsulfonimidoyl)butanoic acid
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-
0.001
3-acetylpyridine adenine dinucleotide
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-
0.8
azaserine
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-
0.28
D-2-hydroxyglutarate
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-
0.6
D-glutamate
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-
0.3
D-glutamine
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-
0.015
DL-ethionine sulfone
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-
0.178
DL-homocysteic acid
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-
1.1
glutamate
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-
0.35
Glutarate
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-
0.25
L-2-Hydroxyglutarate
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-
0.1
L-Albizziin
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14
L-asparagine
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-
2.7
L-aspartate
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-
0.6 - 0.7
L-glutamate
0.0036
L-homocysteine sulfonamide
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-
0.005
L-Methionine sulfone
-
-
0.3
L-methionine-SR-sulfoxide
-
-
5
oxaloacetate
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-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
2.66
-
-
8.05
-
NADH-GOGAT II
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 7.5
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glutamine-dependent NADH oxidase activity
7.2
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broad pH profile with optimum at pH 7.2
7.5 - 8.5
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8
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NADH-GOGAT I, broad pH-optimum centering at pH 8
9
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ammonia-dependent NADH oxidase activity, rate steadily increases from pH 6 to 9
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 9
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at pH 6 and 9 about 50% of activity maximum
7 - 9
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below and above activity declines rapidly
7.2 - 8.2
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optimal pH-range with maximum at pH 7.5
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.1
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NADH-GOGAT I, estimated from amino acid sequence
6.18
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NADH-GOGAT II, estimated from amino acid sequence
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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enzyme activity is detectable in flowers
Manually annotated by BRENDA team
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shorter than 10 mm, supply with 1 mM ammonium ions for 24 h increases the enzyme content in the epidermis and exodermis cells of the root surface
Manually annotated by BRENDA team
additional information
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
194000
-
gel filtration
200000
-
NADH-GOGAT II, gel filtration
222000
-
gel filtration
229000
-
calculated from the cDNA sequence, without N-terminal presequence of 101 amino acids
235000
236000
-
native PAGE
240000
241000
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NADH-GOGAT I and II, estimated from amino acid sequence
265000
590000
-
gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
decamer
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2 * 91000 + 2 * 86000 + 2 * 68000 + 2 * 31000 + 2 * 17500, SDS-PAGE
dimer
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1 * 169000 + 1 * 61000, SDS-PAGE in presence of 1% 2-mercaptoethanol
monomer
additional information
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protein contains domains that correspond to both the large alpha subunit with 46% identity and the small beta subunit with 38% identity of Escherichia coli NADPH-GOGAT, a 57 amino acid highly charged domain connects the two regions homologous to the prokaryotic subunits
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
proteolytic modification
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
35
-
enzyme loses activity after incubation for 10 min
50
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half-life: 10 min, presence of 2-oxoglutarate and L-glutamine stabilizes against thermal denaturation, whereas preincubation with NADH or L-glutamate causes more heat sensitivity
additional information
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
2-mercaptoethanol stabilizes
2-oxoglutarate, 2-mercaptoethanol and EDTA stabilize and have a synergystic effect
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25% glycerol stabilizes
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dithiothreitol stabilizes
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EDTA stabilizes
high levels of sucrose stabilize
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highly labile enzyme activity
instability of enzyme during purification process
-
phenylmethylsulphonyl fluoride is used as precaution against proteolysis
sodium dithionite and glutamine stabilize
-
OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
4C, a few days under air, stable
-
391484
sensitive to oxygen
-
392019
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-80C, 50% glycerol, 100 mM potassium phosphate, 3 mM DTT, stable for up to 1 month
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25C, 4C or -20C, relatively unstable
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4C, a few days under air, stable
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phosphate buffer, per week, 20% loss of activity
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Tris buffer, overnight dialysis, total loss of activity
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
136fold purification
-
150fold partial purification, strain am(132)
-
167fold purification
-
2 isoenzymes: NADH-GOGAT I with 877fold purification and NADH-GOGAT II with 631fold purification
-
208fold purification
-
20fold partial purification
-
350fold purification
-
500fold purification
-
58fold purification
-
74fold partial purification
-
7500fold purification
-
815fold purification
-
partial purification
-
Sephadex G-25 gel filtration
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
full-length cDNA clone and genomic clone for NADH-GOGAT gene
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gene encoding NADH-GltS is cloned, N-terminal three quarters of the gene product is similar to the bacterial alpha subunit from NADPH-GltS
genes gltA and gltA2, phylogenetic analysis
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isolation and characterization of NADH-GOGAT gene with 22 exons and 21 introns, isolation of 7.2 kbp cDNA encoding the complete enzyme
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locus of structural gene encoding NADH-GOGAT is mapped on chromosome 1, detection of quantitative trait loci associated with the enzyme content in leaves
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NADH-GOGAT1, DNA and amino acid sequence determination and analysis, phylogenetic analysis, relative real-time RT-PCR expression analysis; NADH-GOGAT2, DNA and amino acid sequence determination and analysis, phylogenetic analysis, relative real-time RT-PCR expression analysis
open reading frames encoding proteins similar to NADH-GltS are found during sequencing of genome
overexpression of chimeric NADH-GOGAT gene from Medicago sativa fused to the CaMV 35S promoter in transgenic Nicotiana tabacum
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single copy gene glt1, expression pattern analysis
transgenic plants expressing antisense Fd-GOGAT mRNA, NADH-GOGAT activity remains low
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transgenic tobacco lines harboring the GLU1 promoter of the Arabidopsis Fd-GOGAT fused to a GUS reporter gene, NADH-GOGAT activity decreases in old leaves by 32% to 35%
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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possible relationship between NADH-GOGAT, IDH and GDH, IDH is a good candidate to generate the 2-oxoglutarate required for NH4+-induced NADH-GOGAT activity in rice roots, GDH shows no significant differences in its localization between the conditions with or without NH4+ application
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