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Information on EC 1.4.1.13 - glutamate synthase (NADPH) and Organism(s) Azospirillum brasilense and UniProt Accession Q05756

for references in articles please use BRENDA:EC1.4.1.13
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EC Tree
IUBMB Comments
Binds FMN, FAD, 2 [4Fe-4S] clusters and 1 [3Fe-4S] cluster. The reaction takes place in the direction of L-glutamate production. The protein is composed of two subunits, alpha and beta. The alpha subunit is composed of two domains, one hydrolysing L-glutamine to NH3 and L-glutamate (cf. EC 3.5.1.2, glutaminase), the other combining the produced NH3 with 2-oxoglutarate to produce a second molecule of L-glutamate (cf. EC 1.4.1.4, glutamate dehydrogenase [NADP+]). The beta subunit transfers electrons from the cosubstrate. The NH3 is channeled within the alpha subunit through a 31 A channel. The chanelling is very efficient and in the intact alpha-beta complex ammonia is produced only within the complex. In the absence of the beta subunit, coupling between the two domains of the alpha subunit is compromised and some ammonium can leak.
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Azospirillum brasilense
UNIPROT: Q05756
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Word Map
The taxonomic range for the selected organisms is: Azospirillum brasilense
The enzyme appears in selected viruses and cellular organisms
Reaction Schemes
hide(Overall reactions are displayed. Show all >>)
Synonyms
glutamate synthase, gogat, nadph-dependent glutamate synthase, nadph-gogat, l-glutamate synthase, ehno2, ehno1, glutamate synthase (nadph), ph0876, ph1873, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
glutamate synthetase (NADP)
-
-
-
-
glutamine amide-2-oxoglutarate aminotransferase (oxidoreductase, NADP)
-
-
-
-
glutamine-ketoglutaric aminotransferase
-
-
-
-
L-glutamate synthase
-
-
-
-
L-glutamate synthetase
-
-
-
-
L-glutamine:2-oxoglutarate aminotransferase, NADPH oxidizing
-
-
-
-
NADPH-dependent glutamate synthase
NADPH-glutamate synthase
-
-
-
-
NADPH-GOGAT
NADPH-linked glutamate synthase
-
-
-
-
synthase, glutamate (reduced nicotinamide adenine dinucleotide phosphate)
-
-
-
-
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
2 L-glutamate + NADP+ = L-glutamine + 2-oxoglutarate + NADPH + H+
show the reaction diagram
mechanism
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
redox reaction
-
-
-
-
oxidation
-
-
-
-
reduction
-
-
-
-
SYSTEMATIC NAME
IUBMB Comments
L-glutamate:NADP+ oxidoreductase (transaminating)
Binds FMN, FAD, 2 [4Fe-4S] clusters and 1 [3Fe-4S] cluster. The reaction takes place in the direction of L-glutamate production. The protein is composed of two subunits, alpha and beta. The alpha subunit is composed of two domains, one hydrolysing L-glutamine to NH3 and L-glutamate (cf. EC 3.5.1.2, glutaminase), the other combining the produced NH3 with 2-oxoglutarate to produce a second molecule of L-glutamate (cf. EC 1.4.1.4, glutamate dehydrogenase [NADP+]). The beta subunit transfers electrons from the cosubstrate. The NH3 is channeled within the alpha subunit through a 31 A channel. The chanelling is very efficient and in the intact alpha-beta complex ammonia is produced only within the complex. In the absence of the beta subunit, coupling between the two domains of the alpha subunit is compromised and some ammonium can leak.
CAS REGISTRY NUMBER
COMMENTARY hide
37213-53-9
-
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
L-glutamine + 2-oxoglutarate + NADPH + H+
L-glutamate + NADP+
show the reaction diagram
-
-
-
?
ferricyanide + NADPH + H+
ferrocyanide + NADP+
show the reaction diagram
-
-
-
-
r
iodonitrotetrazolium + NADPH + H+
reduced iodonitrotetrazolium + NADP+
show the reaction diagram
-
-
-
-
r
L-glutamine + 2-oxoglutarate + acetylpyridine-NADPH + H+
L-glutamate + acetylpyridine-NADP+
show the reaction diagram
-
-
-
-
r
L-glutamine + 2-oxoglutarate + NADPH
L-glutamate + NADP+
show the reaction diagram
-
-
-
-
?
L-glutamine + 2-oxoglutarate + NADPH + H+
L-glutamate + NADP+
show the reaction diagram
L-glutamine + 2-oxoglutarate + thio-NADPH + H+
L-glutamate + thio-NADP+
show the reaction diagram
-
-
-
-
r
menadione + NADPH + H+
menadiol + NADP+
show the reaction diagram
-
-
-
-
r
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
flavin
NADH
-
no activity with NADH
NADPH
[4Fe-4S]-center
the [4Fe-4S]+1,+2 cluster A displays three Cys and one Glu ligands for the Fe atoms. Residues Cys105 and Cys60 are linked to Fe1 and Fe2, respectively and the Fe3 atom shows bidentate coordination to Glu125 carboxylate, Fe4 is coordinated to Cys99, whose Calpha atom falls 5 A from FAD dimethyl-benzene ring C8 methyl. All Cys ligands to the [4Fe-4S]+1,+2 cluster B (Cys48, Cys51, Cys56, Cys109) are comprised in small subunit GltD loops at the interface with GltB
additional information
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2',5'-diphosphoadenylic acid
-
competitive inhibitor with respect to NADPH
2'-phosphoadenosine-5'-diphospho-5'-beta-D-ribose
2'-phosphoadenylic acid
-
competitive inhibitor with respect to NADPH
3-Aminopyridine adenine dinucleotide phosphate
6-diazo-5-oxo-L-norleucine
-
potent inhibitor, complete loss of activity at 0.1 mM after 10 min of preincubation
acetyl-NADP+
-
-
D-glutamate
-
competitive inhibitor with respect to 2-oxoglutarate, noncompetitive with L-glutamine and uncompetitive with NADPH
iodonitrotetrazolium
-
inhibitor of the L-glutamate:iodonitrotetrazolim oxidoreductase activity of the alpha subunit at concentrations above 0.1 mM
L-asparagine
-
activity decreases to about 0.5% of the original at 25 mM
L-methionine
L-methionine sulfone
-
reversible inhibitor
NADP+
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.23 - 0.75
2-oxoglutarate
0.69 - 2.1
L-glutamine
0.175 - 0.245
NADPH
0.029
2-oxoglutarate
-
-
0.017
acetylpyridine-NADPH
-
in the presence of 2.5 mM 2-oxo-glutarate and 5 mM L-glutamine, the apparent maximal velocity is 3.7% that obtained in the presence of NADPH
0.14
ferricyanide
-
of NADPH: acceptor oxidoreductase activity of the beta subunit of the enzyme
0.05
iodonitrotetrazolium
-
of NADPH: acceptor oxidoreductase activity of the beta subunit of the enzyme
0.23 - 1.5
L-glutamine
0.035
menadione
-
of NADPH: acceptor oxidoreductase activity of the beta subunit of the enzyme
0.0035 - 0.084
NADPH
0.01
thio-NADPH
-
in the presence of 2.5 mM 2-oxo-glutarate and 5 mM L-glutamine, the apparent maximal velocity is 54% that obtained in the presence of NADPH
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
39.5 - 41.9
ferricyanide
20 - 32
iodonitrotetrazolium
25.3
menadione
-
of NADPH:acceptor oxidoreductase acitivity of the beta subunit of the enzyme
18.3 - 30.8
NADPH
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.048
2',5'-diphosphoadenylic acid
-
-
0.0028
2'-phosphoadenosine 5'-diphospho-5-beta-D-ribose
-
-
0.021 - 0.495
2'-phosphoadenosine-5'-diphospho-5'-beta-D-ribose
0.0227 - 0.233
2'-phosphoadenosine-5'-diphosphoribose
0.186
2'-phosphoadenylic acid
-
-
0.0024 - 0.128
3-Aminopyridine adenine dinucleotide phosphate
0.0058
acetyl-NADP+
-
-
0.011 - 0.056
D-glutamate
1.05
L-methionine
-
competitive inhibitor with respect to L-glutamine
0.0059 - 0.037
NADP+
0.00023
thio-NADP+
-
-
additional information
additional information
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
-
-
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION?
GLTD_AZOBR
482
0
52358
Swiss-Prot
-
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
200000
135000
-
4 * 135000 + 4 * 50000, SDS-PAGE
200000
-
approximate value, the alphabeta protomeric form prevails at low protein concentration and at high ionic strength, gel filtration
50000
-
4 * 135000 + 4 * 50000, SDS-PAGE
740000
-
gel filtration
800000
-
approximate value, the tetrameric form is found at high protein concentration and low ionic strength, gel filtration
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
2 * 200000, cryoelectron microscopy and small angle X-ray scattering
homohexamer
6 * 200000, cryoelectron microscopy and small angle X-ray scattering, the hexamer exhibits a concentration-dependent equilibrium with monomers and dimers, in solution the hexamer is destabilized by high ionic strength and to a lower extent by the reaction product NADP+
?
-
x * alpha subunit, 162000, + x * beta subunit, 52300, SDS-PAGE
octamer
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4 * 135000 + 4 * 50000, SDS-PAGE
tetramer
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gel filtration, aggregation state of NADPH-GltS alphabeta holoenzyme
additional information
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
cryo-electron microscopy structures of GltS in oligomeric states alpha4beta3, alpha4beta4, alpha6beta4 and alpha6beta6, in the 3.5- to 4.1 A resolution range
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C47A
-
beta subunit, no significant effect on NADPH:iodonitrotetrazolium oxidoreductase activity of subunit, complete loss of activity of enzyme
C50A
-
beta subunit, no significant effect on NADPH:iodonitrotetrazolium oxidoreductase activity of subunit, complete loss of activity of enzyme
C55A
-
beta subunit, no significant effect on NADPH:iodonitrotetrazolium oxidoreductase activity of subunit, complete loss of activity of enzyme
C59A
-
beta subunit, no significant effect on NADPH:iodonitrotetrazolium oxidoreductase activity of subunit, complete loss of activity of enzyme
G298A
-
mutant with an approximately 10fold decrease of the affinity of the enzyme for pyridine nucleotides with little or no effect on the rate of the enzyme reduction by NADPH, maintains the ability to bind NADPH and FAD, is catalytically active in the NADPH:iodonitrotetrazolium oxidoreductase reaction and has a monomeric state, mutation leads to production of insoluble protein under conditions that yield large amounts of soluble wild-type enzyme and to production of smaller amounts of enzyme
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.3 - 8.3
-
-
391446
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
of the alpha subunit, using column chromatography on Q-Sepharose and Ultrogel AcA34
-
of the G298A-beta subunit, using column chromatography on Reactive Red or Amicon Red resins and gel filtration on Ultrogel AcA 34
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of the recombinant enzyme beta subunit, using ammonium sulfate fractionation, affinity chromatography on Reactive Red, ultrafiltration and column chromatography on Ultrogel AcA 54
-
of the recombinant enzyme from overproducing Escherichia coli cells, using ion exchange chromatography on Q-Sepharose, gel filtration on Sephacryl S-300 and affinity chromatography on 2',5' ADP-Sepharose 4B colum
-
recombuinant enzyme and isolated beta subunit
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using heat treatment, ammonium sulfate precipitation, column chromatography on DEAE-Trisacryl, gel filtration and affinity chromatogray
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21 (DE3) cells
expression of the holoenzyme in Escherichia coli
-
overproduction of the holoenzyme and of the alpha subunit in Escherichia coli
-
production of the alpha and beta subunits in Escherichia coli
-
production of the beta subunit in Escherichia coli
-
production of the G298A-beta subunit in Escherichia coli
-
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Vanoni, M.A.; Edmondson, D.E.; Zanetti, G.; Curti, B.
Characterization of the flavins and the iron-sulfur centers of glutamate synthase from Azospirillum brasilense by absorption, circular dichroism, and electron paramagnetic resonance spectroscopies
Biochemistry
31
4613-4623
1992
Azospirillum brasilense
Manually annotated by BRENDA team
Vanoni, M.A.; Edmondson, D.E.; Rescigno, M.; Zanetti, G.; Curti, B.
Mechanistic studies on Azospirillum brasilense glutamate synthase
Biochemistry
30
11478-11484
1991
Azospirillum brasilense
Manually annotated by BRENDA team
Ratti, S.; Curti, B.; Zanetti, G.; Galli, E.
Purification and characterization of glutamate synthase from Azospirillum brasilense
J. Bacteriol.
163
724-729
1985
Azospirillum brasilense
Manually annotated by BRENDA team
Vanoni, M.A.; Verzotti, E.; Zanetti, G.; Curti, B.
Properties of the recombinant beta subunit of glutamate synthase
Eur. J. Biochem.
236
937-946
1996
Azospirillum brasilense
Manually annotated by BRENDA team
Vanoni, M.A.; Fischer, F.; Ravasio, S.; Verzotti, E.; Edmondson, D.E.; Hagen, W.R.; Zanetti, G.; Curti, B.
The recombinant alpha subunit of glutamate synthase: spectroscopic and catalytic properties
Biochemistry
37
1828-1838
1998
Azospirillum brasilense
Manually annotated by BRENDA team
Morandi, P.; Valzasina, B.; Colombo, C.; Curti, B.; Vanoni, M.A.
Glutamate Synthase: Identification of the NADPH-Binding Site by Site-Directed Mutagenesis
Biochemistry
39
727-735
2000
Azospirillum brasilense
Manually annotated by BRENDA team
Stabile, H.; Curti, B.; Vanoni, M.A.
Functional properties of recombinant Azospirillum brasilense glutamate synthase, a complex iron-sulfur flavoprotein
Eur. J. Biochem.
267
2720-2730
2000
Azospirillum brasilense
Manually annotated by BRENDA team
Ravasio, S.; Curti, B.; Vanoni, M.A.
Determination of the Midpoint Potential of the FAD and FMN Flavin Cofactors and of the 3Fe-4S Cluster of Glutamate Synthase
Biochemistry
40
5533-5541
2001
Azospirillum brasilense
Manually annotated by BRENDA team
Ravasio, S.; Dossena, L.; Martin-Figueroa, E.; Florencio, F.J.; Mattevi, A.; Morandi, P.; Curti, B.; Vanoni, M.A.
Properties of the recombinant ferredoxin-dependent glutamate synthase of Synechocystis PCC6803. Comparison with the Azospirillum brasilense NADPH-dependent enzyme and its isolated alpha subunit
Biochemistry
41
8120-8133
2002
Azospirillum brasilense
Manually annotated by BRENDA team
Agnelli, P.; Dossena, L.; Colombi, P.; Mulazzi, S.; Morandi, P.; Tedeschi, G.; Negri, A.; Curti, B.; Vanoni, M.A.
The unexpected structural role of glutamate synthase [4Fe-4S](+1,+2) clusters as demonstrated by site-directed mutagenesis of conserved C residues at the N-terminus of the enzyme beta subunit
Arch. Biochem. Biophys.
436
355-366
2005
Azospirillum brasilense
Manually annotated by BRENDA team
Petoukhov, M.V.; Svergun, D.I.; Konarev, P.V.; Ravasio, S.; van den Heuvel, R.H.; Curti, B.; Vanoni, M.A.
Quaternary structure of Azospirillum brasilense NADPH-dependent glutamate synthase in solution as revealed by synchrotron radiation x-ray scattering
J. Biol. Chem.
278
29933-29939
2003
Azospirillum brasilense (Q05755), Azospirillum brasilense
Manually annotated by BRENDA team
Coiro, V.M.; Di Nola, A.; Vanoni, M.A.; Aschi, M.; Coda, A.; Curti, B.; Roccatano, D.
Molecular dynamics simulation of the interaction between the complex iron-sulfur flavoprotein glutamate synthase and its substrates
Protein Sci.
13
2979-2991
2004
Azospirillum brasilense
Manually annotated by BRENDA team
Suzuki, A.; Knaff, D.B.
Glutamate synthase: structural, mechanistic and regulatory properties, and role in the amino acid metabolism
Photosynth. Res.
83
191-217
2005
Acidithiobacillus ferrooxidans (Q56266), Acidithiobacillus ferrooxidans (Q56267), Archaeoglobus fulgidus (O29309), Azospirillum brasilense (Q05755), Azospirillum brasilense (Q05756), Azospirillum brasilense (Q59084), Bacillus subtilis (P39812), Escherichia coli (P09831), Escherichia coli (P09832), Klebsiella aerogenes, Methanocaldococcus jannaschii (Q58746), Methanococcus thermoautotrophicum (O26308), no activity in Arabidopsis thaliana, Priestia megaterium, Pseudomonas aeruginosa (P95456), Pseudomonas aeruginosa (P95457), Pyrococcus sp., Pyrococcus sp. KOD1, Rhizobium etli (Q9ZFB8), Rhizobium etli (Q9ZFB9), Rhodospirillum rubrum, Salmonella enterica subsp. enterica serovar Typhimurium (Q8ZLR3), Salmonella enterica subsp. enterica serovar Typhimurium (Q8ZLR4)
Manually annotated by BRENDA team
Vanoni, M.A.; Dossena, L.; van den Heuvel, R.H.; Curti, B.
Structure-function studies on the complex iron-sulfur flavoprotein glutamate synthase: the key enzyme of ammonia assimilation
Photosynth. Res.
83
219-238
2005
Azospirillum brasilense
Manually annotated by BRENDA team
Cottevieille, M.; Larquet, E.; Jonic, S.; Petoukhov, M.V.; Caprini, G.; Paravisi, S.; Svergun, D.I.; Vanoni, M.A.; Boisset, N.
The subnanometer resolution structure of the glutamate synthase 1.2-MDa hexamer by cryoelectron microscopy and its oligomerization behavior in solution: functional implications
J. Biol. Chem.
283
8237-8249
2008
Azospirillum brasilense (Q05756)
Manually annotated by BRENDA team
Swuec, P.; Chaves-Sanjuan, A.; Camilloni, C.; Vanoni, M.A.; Bolognesi, M.
Cryo-EM structures of Azospirillum brasilense glutamate synthase in its oligomeric assemblies
J. Mol. Biol.
431
4523-4526
2019
Azospirillum brasilense (Q05755 and Q05756), Azospirillum brasilense
Manually annotated by BRENDA team