Glutamate synthase (NADPH)

 

Binds FMN, FAD, 2 [4Fe-4S] clusters and 1 [3Fe-4S] cluster. The protein is composed of two subunits, alpha and beta. The alpha subunit is composed of two domains, one hydrolysing L-glutamine to ammonia and L-glutamate, the other combining the produced ammonia with 2-oxoglutarate to produce a second molecule of L-glutamate (cf. EC 1.4.1.4, glutamate dehydrogenase [NADP+]). The ammonia is channelled through a 31 Å channel in the active protein. This entry represents the alpha subunit reaction.

Glutamine hydrolysis is absent (or negligible) in the absence of the other substrates. The enzyme also catalyses the oxidation of NADPH, the ammonia-dependent synthesis of glutamate from 2-oxoglutarate and ammonia and the oxidation of L-glutamate in the presence of iodonitrotetrazolium salts, the latter two of which only occur at high pH. There is still much to be determined with respect to the mechanism of this enzyme [PMID:18421771] as well as how the cross-control of the synthase and glutaminase activities occurs with such precision. This enzyme utilises three iron-sulfur clusters (two Fe4S4 and one Fe3S4) as well as FMN and FAD as cofactors.

 

Reference Protein and Structure

Sequence
Q05755 UniProt (1.4.1.13) IPR029055 (Sequence Homologues) (PDB Homologues)
Biological species
Azospirillum brasilense (Bacteria) Uniprot
PDB
1ea0 - Alpha subunit of A. brasilense glutamate synthase (3.0 Å) PDBe PDBsum 1ea0
Catalytic CATH Domains
3.60.20.10 CATHdb 3.20.20.70 CATHdb (see all for 1ea0)
Cofactors
Fmnh2(2-) (1), Fadh2(2-) (1), Tri-mu-sulfido-mu3-sulfido-triiron(0) (1), Tetra-mu3-sulfido-tetrairon (2) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:1.4.1.13)

L-glutamine zwitterion
CHEBI:58359ChEBI
+
hydron
CHEBI:15378ChEBI
+
2-oxoglutarate(2-)
CHEBI:16810ChEBI
+
NADPH(4-)
CHEBI:57783ChEBI
→
NADP(3-)
CHEBI:58349ChEBI
+
L-glutamate(1-)
CHEBI:29985ChEBI
Alternative enzyme names: L-glutamate synthase, L-glutamate synthetase, L-glutamine:2-oxoglutarate aminotransferase, NADPH oxidizing, NADPH-dependent glutamate synthase, NADPH-glutamate synthase, NADPH-linked glutamate synthase, Glutamate (reduced nicotinamide adenine dinucleotide phosphate) synthase, Glutamate synthetase (NADP), Glutamine amide-2-oxoglutarate aminotransferase (oxidoreductase, NADP), Glutamine-ketoglutaric aminotransferase, GOGAT,

Enzyme Mechanism

Introduction

NADP reduces the FAD cofactor. A single electron is transferred from FAD, via the iron-sulfur clusters and Met479 to the FMN cofactor. A second single electron is transferred from FAD, via the iron-sulfur clusters and Met479 to the FMN cofactor. The N-terminus of Cys1 deprotonates water, which deprotonates the thiol group of Cys1, initiating a nucleophilic attack on the amide carbon in an addition reaction. The oxyanion initiates an elimination that cleaves ammonia from the bound L-glutamine substrate. Ammonia deprotonates water, which deprotonates the N-terminus of Cys1. The N-terminus of Cys1 deprotonates water, which initiates a nucleophilic attack on the carbonyl carbon of the covalently bound intermediate in an addition reaction. The oxyanion initiates an elimination that cleaves the C-S bond, the thiolate of Cys1 deprotonates water, which deprotonates the N-terminus of Cys1. Ammonia initiates a nucleophilic attack on the C2 of 2-oxoglutarate in an addition reaction. Water is produced through an intramolecular elimination forming the 2-iminoglutarate intermediate. FMN donates a hydride to the 2-iminoglutarate intermediate.

Catalytic Residues Roles

UniProt PDB* (1ea0)
Cys37 (N-term) Cys1A (N-term) Acts as a general acid/base, activating its own side chain via a water relay. proton acceptor, proton donor
Glu922 Glu886A It has been suggested that Glu886 acts as a general acid/base in the final protonation step of the reaction. proton relay, proton acceptor, proton donor
Gly268 (main-N), Asn267 Gly232A (main-N), Asn231A Forms the oxyanion hole that stabilises the reactive intermediates and transition states in the glutaminase domain. hydrogen bond donor, electrostatic stabiliser
Cys37 Cys1A Activated by the N-terminus of the protein, this acts as a catalytic nucleophile in the glutaminase domain of the protein. covalently attached, hydrogen bond acceptor, hydrogen bond donor, nucleofuge, proton acceptor, nucleophile, proton donor
Met515 Met479A Forms part of the electron relay chain that transfers single electrons from the FAD to the FMN cofactor. single electron relay, single electron acceptor, single electron donor
Lys973 Lys937A Helps stabilise the reactive intermediates in the synthase domain. electrostatic stabiliser
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

hydride transfer, overall reactant used, cofactor used, overall product formed, intermediate formation, redox reaction, proton transfer, electron relay, native state of cofactor regenerated, bimolecular nucleophilic addition, proton relay, enzyme-substrate complex formation, unimolecular elimination by the conjugate base, enzyme-substrate complex cleavage, deamination, intermediate collapse, intramolecular elimination, native state of enzyme regenerated

References

  1. Vanoni MA et al. (2008), IUBMB Life, 60, 287-300. Structure–function studies of glutamate synthases: A class of self-regulated iron-sulfur flavoenzymes essential for nitrogen assimilation. DOI:10.1002/iub.52. PMID:18421771.
  2. Vanoni MA et al. (2005), Arch Biochem Biophys, 433, 193-211. Structure–function studies on the iron–sulfur flavoenzyme glutamate synthase: an unexpectedly complex self-regulated enzyme. DOI:10.1016/j.abb.2004.08.033. PMID:15581577.
  3. Vanoni MA et al. (2005), Photosynth Res, 83, 219-238. Structure-function studies on the complex iron-sulfur flavoprotein glutamate synthase: the key enzyme of ammonia assimilation. DOI:10.1007/s11120-004-2438-z. PMID:16143853.
  4. van den Heuvel RH et al. (2003), J Mol Biol, 330, 113-128. The Active Conformation of Glutamate Synthase and its Binding to Ferredoxin. DOI:10.1016/s0022-2836(03)00522-9. PMID:12818206.
  5. Ravasio S et al. (2001), Biochemistry, 40, 5533-5541. Determination of the Midpoint Potential of the FAD and FMN Flavin Cofactors and of the 3Fe−4S Cluster of Glutamate Synthase†. DOI:10.1021/bi0100889. PMID:11331018.
  6. Binda C et al. (2000), Structure, 8, 1299-1308. Cross-Talk and Ammonia Channeling between Active Centers in the Unexpected Domain Arrangement of Glutamate Synthase. DOI:10.1016/s0969-2126(00)00540-2. PMID:11188694.

Step 1. NADP reduces the FAD cofactor. It is not yet clear exactly how this step occurs and what residues, if any, are involved

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Catalytic Residues Roles

Residue Roles

Chemical Components

hydride transfer, overall reactant used, cofactor used, overall product formed, intermediate formation

Step 2. The FMN is in the synthase domain. A single electron is transferred from FAD, via the iron-sulfur clusters and Met479 to the FMN cofactor. Studies suggest that the 2-oxoglutarate substrate needs to bind before the electron transfer can occur [PMID:11331018]. This is the first of two single electron transfers. The identity of the proton donors/acceptors is currently unclear.

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Catalytic Residues Roles

Residue Roles
Met479A single electron relay, single electron acceptor, single electron donor

Chemical Components

redox reaction, proton transfer, intermediate formation, cofactor used, electron relay

Step 3. The FMN is in the synthase domain. A second single electron is transferred from FAD, via the iron-sulfur clusters and Met479 to the FMN cofactor.

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Catalytic Residues Roles

Residue Roles
Met479A single electron relay, single electron acceptor, single electron donor

Chemical Components

redox reaction, proton transfer, intermediate formation, native state of cofactor regenerated, electron relay

Step 4. This reaction occurs in the glutaminase domain. The N-terminus of Cys1 deprotonates water, which deprotonates the thiol group of Cys1, initiating a nucleophilic attack on the amide carbon in an addition reaction. The N-terminus of Cys1 may also directly deprotonate the thiol group of Cys1 and not involve a water molecule.

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Catalytic Residues Roles

Residue Roles
Cys1A hydrogen bond donor
Gly232A (main-N) hydrogen bond donor, electrostatic stabiliser
Asn231A hydrogen bond donor, electrostatic stabiliser
Cys1A (N-term) proton acceptor
Cys1A nucleophile, proton donor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, proton relay, enzyme-substrate complex formation, overall reactant used, intermediate formation

Step 5. This reaction occurs in the glutaminase domain. The oxyanion initiates an elimination that cleaves ammonia from the bound L-glutamine substrate. Ammonia deprotonates water, which deprotonates the N-terminus of Cys1.

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Catalytic Residues Roles

Residue Roles
Cys1A covalently attached
Gly232A (main-N) hydrogen bond donor, electrostatic stabiliser
Asn231A hydrogen bond donor, electrostatic stabiliser
Cys1A (N-term) proton donor

Chemical Components

proton transfer, ingold: unimolecular elimination by the conjugate base, proton relay, intermediate formation, enzyme-substrate complex cleavage, deamination

Step 6. This reaction occurs in the glutaminase domain. The N-terminus of Cys1 deprotonates water, which initiates a nucleophilic attack on the carbonyl carbon of the covalently bound intermediate in an addition reaction.

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Catalytic Residues Roles

Residue Roles
Cys1A covalently attached
Gly232A (main-N) hydrogen bond donor, electrostatic stabiliser
Asn231A hydrogen bond donor, electrostatic stabiliser
Cys1A (N-term) proton acceptor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, enzyme-substrate complex formation, overall reactant used, intermediate formation

Step 7. This reaction occurs in the glutaminase domain. The oxyanion initiates an elimination that cleaves the C-S bond, the thiolate of Cys1 deprotonates water, which deprotonates the N-terminus of Cys1.

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Catalytic Residues Roles

Residue Roles
Cys1A hydrogen bond acceptor
Gly232A (main-N) hydrogen bond donor, electrostatic stabiliser
Asn231A hydrogen bond donor, electrostatic stabiliser
Cys1A nucleofuge, proton acceptor
Cys1A (N-term) proton donor

Chemical Components

proton transfer, ingold: unimolecular elimination by the conjugate base, proton relay, intermediate collapse, overall product formed, enzyme-substrate complex cleavage

Step 8. This reaction occurs in the synthase domain. Ammonia initiates a nucleophilic attack on the C2 of 2-oxoglutarate in an addition reaction.

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Catalytic Residues Roles

Residue Roles
Lys937A electrostatic stabiliser

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, intermediate formation, overall reactant used

Step 9. This reaction occurs in the synthase domain. Water is produced through an intramolecular elimination forming the 2-iminoglutarate intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys937A electrostatic stabiliser

Chemical Components

proton transfer, ingold: intramolecular elimination, intermediate formation, overall reactant used

Step 10. This reaction occurs in the synthase domain. FMN donates a hydride to the 2-iminoglutarate intermediate. The observation of reduction of FMN by the L-glutamate product without the formation of a semiquinone species suggests that the 2-iminoglutarate reduction occurs by direct hydride transfer from the reduced FMN cofactor [PMID:18421771]. It is unclear where the proton comes from, one suggestion is Glu886, although it has also been suggested that the ammonia channel is filled by water, and so water could act as the general acid [PMID:18421771].

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys937A electrostatic stabiliser
Glu886A proton acceptor, proton donor, proton relay

Chemical Components

proton transfer, hydride transfer, ingold: unimolecular elimination by the conjugate base, ingold: bimolecular nucleophilic addition, native state of cofactor regenerated, overall product formed, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. NADP reduces the FAD cofactor. It is not yet clear exactly how this step occurs and what residues, if any, are involved

Step 2. The FMN is in the synthase domain. A single electron is transferred from FAD, via the iron-sulfur clusters and Met479 to the FMN cofactor. Studies suggest that the 2-oxoglutarate substrate needs to bind before the electron transfer can occur [PMID:11331018]. This is the first of two single electron transfers. The identity of the proton donors/acceptors is currently unclear.

Step 3. The FMN is in the synthase domain. A second single electron is transferred from FAD, via the iron-sulfur clusters and Met479 to the FMN cofactor.

Step 4. This reaction occurs in the glutaminase domain. The N-terminus of Cys1 deprotonates water, which deprotonates the thiol group of Cys1, initiating a nucleophilic attack on the amide carbon in an addition reaction. The N-terminus of Cys1 may also directly deprotonate the thiol group of Cys1 and not involve a water molecule.

Step 5. This reaction occurs in the glutaminase domain. The oxyanion initiates an elimination that cleaves ammonia from the bound L-glutamine substrate. Ammonia deprotonates water, which deprotonates the N-terminus of Cys1.

Step 6. This reaction occurs in the glutaminase domain. The N-terminus of Cys1 deprotonates water, which initiates a nucleophilic attack on the carbonyl carbon of the covalently bound intermediate in an addition reaction.

Step 7. This reaction occurs in the glutaminase domain. The oxyanion initiates an elimination that cleaves the C-S bond, the thiolate of Cys1 deprotonates water, which deprotonates the N-terminus of Cys1.

Step 8. This reaction occurs in the synthase domain. Ammonia initiates a nucleophilic attack on the C2 of 2-oxoglutarate in an addition reaction.

Step 9. This reaction occurs in the synthase domain. Water is produced through an intramolecular elimination forming the 2-iminoglutarate intermediate.

Step 10. This reaction occurs in the synthase domain. FMN donates a hydride to the 2-iminoglutarate intermediate. The observation of reduction of FMN by the L-glutamate product without the formation of a semiquinone species suggests that the 2-iminoglutarate reduction occurs by direct hydride transfer from the reduced FMN cofactor [PMID:18421771]. It is unclear where the proton comes from, one suggestion is Glu886, although it has also been suggested that the ammonia channel is filled by water, and so water could act as the general acid [PMID:18421771].

Products.

Contributors

Gemma L. Holliday, Amelia Brasnett