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L-glutamine + 2-oxoglutarate + NADPH + H+
L-glutamate + NADP+
-
-
-
?
ferricyanide + NADPH + H+
ferrocyanide + NADP+
-
-
-
-
r
iodonitrotetrazolium + NADPH + H+
reduced iodonitrotetrazolium + NADP+
-
-
-
-
r
L-glutamine + 2-oxoglutarate + acetylpyridine-NADPH + H+
L-glutamate + acetylpyridine-NADP+
-
-
-
-
r
L-glutamine + 2-oxoglutarate + NADPH
L-glutamate + NADP+
-
-
-
-
?
L-glutamine + 2-oxoglutarate + NADPH + H+
L-glutamate + NADP+
L-glutamine + 2-oxoglutarate + thio-NADPH + H+
L-glutamate + thio-NADP+
-
-
-
-
r
menadione + NADPH + H+
menadiol + NADP+
-
-
-
-
r
additional information
?
-
L-glutamine + 2-oxoglutarate + NADPH + H+
L-glutamate + NADP+
-
-
-
?
L-glutamine + 2-oxoglutarate + NADPH + H+
L-glutamate + NADP+
-
-
-
?
L-glutamine + 2-oxoglutarate + NADPH + H+
L-glutamate + NADP+
-
-
-
?
L-glutamine + 2-oxoglutarate + NADPH + H+
L-glutamate + NADP+
-
-
-
?
L-glutamine + 2-oxoglutarate + NADPH + H+
L-glutamate + NADP+
-
-
-
?
L-glutamine + 2-oxoglutarate + NADPH + H+
L-glutamate + NADP+
-
-
-
?
L-glutamine + 2-oxoglutarate + NADPH + H+
L-glutamate + NADP+
-
-
-
ir
L-glutamine + 2-oxoglutarate + NADPH + H+
L-glutamate + NADP+
-
when L-glutamine is replaced by ammonia as the amino-group donor, the catalytic activity is less than 1%
-
?
L-glutamine + 2-oxoglutarate + NADPH + H+
L-glutamate + NADP+
-
2-oxoglutarate promotes electron transfer from FAD to 3Fe-4S cluster of the holoenzyme
-
?
additional information
?
-
-
the enzyme beta subunit is devoid of glutamate synthase activity in either direction at both pH 7.5 and 9.5, but it can oxidize NADPH and transfer electrons to synthetic electron acceptors like iodonitrotetrazolium, ferricyanide, menadione, dichloroindophenol, the beta subunit is highly specific toward NADPH, the rate of oxidation of NADH in the presence of electron acceptors is less than 5% of that measured with NADPH
-
-
?
additional information
?
-
-
the recombinant enzyme has diaphorase activity, it can oxidize NADPH and transfer electrons to synthetic electron acceptors like iodonitrotetrazolium and ferricyanide
-
-
?
additional information
?
-
-
under conditions of physiological pH the enzyme exhibits a reversible half-reaction, but overall catalysis is essentially irreversible
-
-
?
additional information
?
-
-
the alpha subunit catalyzes the synthesis of glutamate from L-glutamine and 2-oxoglutarate, provided that a reducing system is present, reducing system: dithionite and methyl viologen
-
-
?
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NADH
-
no activity with NADH
[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
FAD
-
-
FAD
the isoalloxazine ring is almost solvent inaccessible, neighboring residues are Ile98, Pro100, Leu186, Ile191, Lys195, Leu266, Asp300, Thr301, Asp304, Leu450 and Val451, together with backbone atoms of the surrounding regions
flavin
-
flavoenzyme
flavin
-
contains FAD and FMN
flavin
-
0.86 mol FAD per mol of mutant beta subunit, 0.83 mol FAD per mol of the wild type species
flavin
-
the alpha subunit contains FMN as flavin cofactor, 0.94 FMN bound per alpha subunit
flavin
-
0.83 mol FAD and 0.86 mol FMN per mol alphabeta protomer
flavin
-
ratio FAD: FMN is 1
flavin
-
the beta subunit contains the binding site for FAD, 0.83 mol FAD per mol beta subunit
NADPH
-
-
NADPH
-
oxidizes NADPH stereospecifically at the 4S position
NADPH
-
the C-terminal potential ADP-binding fold of the beta subunit is the NADPH-binding site of the enzyme
NADPH
-
the beta subunit contains the NADPH binding site of the enzyme
additional information
-
the enzyme contains three distinct ion-sulfur centers per alphabeta protomer
-
additional information
-
the enzyme contains three different iron-sulfur clusters, one 3Fe-4S center on the alpha subunit and two 4Fe-4S clusters of unknown location, 11.7 mol sulfur per mol alphabeta protomer
-
additional information
-
the alpha subunit contains the [3Fe-4S] cluster of the enzyme
-
additional information
-
midpoint potential value of the FMN cofactor: approximately -240 mV, midpoint potential value of the 3Fe-4S cluster: approximately -270 mV, midpoint potential value of the FAD cofactor: approximately -340 mV for the beta subunit and -300 mV for the holoenzyme
-
additional information
-
the FMN and FAD prosthetic groups are demonstrated to be nonequivalent with respect to their reactivities with sulfite, sulfite reacts with only one of the two flavins forming an N(5)-sulfite adduct
-
additional information
-
thio-NADPH and acetylpyridine-NADPH can be used as electron donors but are less efficient than NADPH
-
additional information
-
the enzyme contains 7.9 sulfur atoms per protomer with a molecular weight of 185000
-
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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
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 sulfone
-
reversible inhibitor
2'-phosphoadenosine-5'-diphospho-5'-beta-D-ribose
-
-
2'-phosphoadenosine-5'-diphospho-5'-beta-D-ribose
-
inhibitor of the NADPH:iodonitrotetrazolium oxidoreductase reaction of the G298A-beta subunit, competitive with respect to NADPH
2'-phosphoadenosine-5'-diphospho-5'-beta-D-ribose
-
for the NADPH: acceptor oxidoreductase activity of the beta subunit of the enzyme, uncompetitive inhibition with ferricyanide or iodonitrotetrazolium as substrate, competitive inhibition with NADPH as substrate
3-Aminopyridine adenine dinucleotide phosphate
-
-
3-Aminopyridine adenine dinucleotide phosphate
-
inhibitor of the NADPH:iodonitrotetrazolium oxidoreductase reaction of the G298A-beta subunit, competitive with respect to NADPH
3-Aminopyridine adenine dinucleotide phosphate
-
good inhibitor of the holoenzyme, competitive with respect to NADPH
L-methionine
-
-
L-methionine
-
competitive inhibitor with respect to L-glutamine
NADP+
-
-
NADP+
-
inhibitor of the NADPH:iodonitrotetrazolium oxidoreductase reaction of the G298A-beta subunit, competitive with respect to NADPH
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0.23 - 0.75
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
0.035
menadione
-
of NADPH: acceptor oxidoreductase activity of the beta subunit of the enzyme
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
0.23
2-oxoglutarate
dimeric enzyme, at 25°C in 50 mM HEPES/KOH buffer, pH 7.5, 1 M NaCl, in the presence of 10 mM L-glutamine, and 0.1 mM NADPH
0.75
2-oxoglutarate
hexameric enzyme, at 25°C in 50 mM HEPES/KOH buffer, pH 7.5, 1 M NaCl, in the presence of 10 mM L-glutamine, and 0.1 mM NADPH
0.69
L-glutamine
dimeric enzyme, at 25°C in 50 mM HEPES/KOH buffer, pH 7.5, 1 M NaCl, in the presence of 2.5 mM 2-oxoglutarate, and 0.1 mM NADPH
2.1
L-glutamine
hexameric enzyme, at 25°C in 50 mM HEPES/KOH buffer, pH 7.5, 1 M NaCl, in the presence of 2.5 mM 2-oxoglutarate, and 0.1 mM NADPH
0.175
NADPH
dimeric enzyme, at 25°C in 50 mM HEPES/KOH buffer, pH 7.5, 1 M NaCl, in the presence of 10 mM L-glutamine, and 5 mM 2-oxoglutarate
0.245
NADPH
hexameric enzyme, at 25°C in 50 mM HEPES/KOH buffer, pH 7.5, 1 M NaCl, in the presence of 10 mM L-glutamine, and 5 mM 2-oxoglutarate
0.23
L-glutamine
-
holoenzyme, buffer: Hepes, pH 8.5 or 7.5
0.63
L-glutamine
-
alpha subunit, buffer: Hepes, pH 7.5
0.73
L-glutamine
-
holoenzyme, buffer: CAPS, pH 9.5
0.92
L-glutamine
-
alpha subunit, buffer: Hepes, pH 8.5
1.5
L-glutamine
-
alpha subunit, buffer: CAPS, pH 9.5
0.0035
NADPH
-
of NADPH: acceptor oxidoreductase activity of the beta subunit of the enzyme, acceptor: iodonitrotetrazolium
0.0098
NADPH
-
of NADPH: acceptor oxidoreductase activity of the beta subunit of the enzyme, acceptor: menadione
0.0118
NADPH
-
of NADPH: acceptor oxidoreductase activity of the beta subunit of the enzyme, acceptor: ferricyanide
0.084
NADPH
-
of NADPH: iodonitrotetrazolium oxidoreductase activity of the G298A-beta subunit
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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
0.011 - 0.056
D-glutamate
1.05
L-methionine
-
competitive inhibitor with respect to L-glutamine
additional information
additional information
-
0.021
2'-phosphoadenosine-5'-diphospho-5'-beta-D-ribose
-
for the NADPH:acceptor oxidoreductase activity of the beta subunit of the enzyme, substrate: NADPH, cosubstrate: ferricyanide
0.08
2'-phosphoadenosine-5'-diphospho-5'-beta-D-ribose
-
for the NADPH:acceptor oxidoreductase activity of the beta subunit of the enzyme, substrate: NADPH, cosubstrate: iodonitrotetrazolium
0.18
2'-phosphoadenosine-5'-diphospho-5'-beta-D-ribose
-
for the NADPH:acceptor oxidoreductase activity of the beta subunit of the enzyme, substrate: ferricyanide, cosubstrate: NADPH
0.495
2'-phosphoadenosine-5'-diphospho-5'-beta-D-ribose
-
for the NADPH:acceptor oxidoreductase activity of the beta subunit of the enzyme, substrate: iodonitrotetrazolium, cosubstrate: NADPH
0.0227
2'-phosphoadenosine-5'-diphosphoribose
-
inhibitor of the NADPH: iodonitrotetrazolium oxidoreductase reaction of wild type-beta subunit
0.233
2'-phosphoadenosine-5'-diphosphoribose
-
inhibitor of the NADPH: iodonitrotetrazolium oxidoreductase reaction of G298A-beta subunit
0.0024
3-Aminopyridine adenine dinucleotide phosphate
-
-
0.0024
3-Aminopyridine adenine dinucleotide phosphate
-
inhibitor of the holoenzyme, competitive with NADPH
0.0112
3-Aminopyridine adenine dinucleotide phosphate
-
inhibitor of the NADPH: iodonitrotetrazolium oxidoreductase reaction of wild type-beta subunit
0.128
3-Aminopyridine adenine dinucleotide phosphate
-
inhibitor of the NADPH: iodonitrotetrazolium oxidoreductase reaction of G298A-beta subunit
0.011
D-glutamate
-
good competitive inhibitor with respect to 2-oxoglutarate
0.044
D-glutamate
-
uncompetitive inhibitor with NADPH
0.056
D-glutamate
-
noncompetitive inhibitor with L-glutamine
0.0059
NADP+
-
-
0.037
NADP+
-
inhibitor of the NADPH: iodonitrotetrazolium oxidoreductase reaction of G298A-beta subunit
additional information
additional information
-
-
-
additional information
additional information
-
-
-
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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
-
4 * 135000 + 4 * 50000, SDS-PAGE
tetramer
-
gel filtration, aggregation state of NADPH-GltS alphabeta holoenzyme
additional information
analysis by synchrotron radiation x-ray solution scattering, alpha subunit and alphabeta holoenzyme are tetrameric in solution, beta subunit is a mixture of monomers and dimers. The (alphabeta)4 holoenzyme is similar to the tetrameric alpha4 complex with the beta subunits occupying the periphery, thus allowing independent catalytic activities of the alphabeta protomers
additional information
-
analysis by synchrotron radiation x-ray solution scattering, alpha subunit and alphabeta holoenzyme are tetrameric in solution, beta subunit is a mixture of monomers and dimers. The (alphabeta)4 holoenzyme is similar to the tetrameric alpha4 complex with the beta subunits occupying the periphery, thus allowing independent catalytic activities of the alphabeta protomers
additional information
-
modelling of unliganded alpha subunit as well as in complex with L-glutamine and 2-oxoglutarate, subunit exists in a catalytically inactive conformation unable to bind glutamine, and in a catalytically competent conformation, which is stabilized by the glutamine substrate. Binding of L-methionine sulfone causes a coordinated rigid-body motion that results in the inactive conformation
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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
brenda
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
brenda
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
brenda
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
brenda
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
brenda
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
brenda
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
brenda
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
brenda
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
brenda
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
brenda
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
brenda
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
brenda
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)
brenda
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
brenda
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)
brenda
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
brenda