Literature summary for 1.4.1.3 extracted from

Dimovasili, C.; Fadouloglou, V.E.; Kefala, A.; Providaki, M.; Kotsifaki, D.; Kanavouras, K.; Sarrou, I.; Plaitakis, A.; Zaganas, I.; Kokkinidis, M.
Crystal structure of glutamate dehydrogenase 2, a positively selected novel human enzyme involved in brain biology and cancer pathophysiology (2021), J. Neurochem., 157, 802-815 .

Activating Compound

Activating Compound	Comment	Organism	Structure
ADP	ADP is an activator of hGDH1 and hGDH2 which binds only to the open state	Homo sapiens
ADP	ADP is an activator of hGDH1 and hGDH2 which binds only to the open state. hGDH1mutant R443S/G456A exhibits a reduced ADP sensitivity compared both to hGDH1 and to hGDH2. The addition of the M415L and R470H mutations results in an even more pronounced drop in ADP affinity	Homo sapiens

Cloned(Commentary)

Cloned (Comment)	Organism
gene GLUD1, recombinant expression of mutant enzymes in Spodoptera frugiperda SF21 cells	Homo sapiens
gene GLUD2, recombinant expression of wild-type enzyme in Spodoptera frugiperda SF21 cells	Homo sapiens

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified isozyme hGDH2, hGDH2 is crystallized in the absence of allosteric regulators or active site ligands, by hanging drop vapour diffusion method, mixing of 0.002 ml of 9.7 mg/ml protein solution with an equal volume of reservoir solution containing 8% PEG 8000, 15% MPD, 0.4 M NaCl and 0.1 M phosphate, pH 7.0, and equilibration against 1.0 ml of reservoir solution at 18°C, method screening and optimization, X-ray diffraction structure determination and analysis at 2.9 A resolution, molecular replacement method and modelling using the hexameric human apo-enzyme hGDH1 structure (PDB ID 1L1F) as a search model	Homo sapiens

Crystallization (Comment)

Organism

purified isozyme hGDH2, hGDH2 is crystallized in the absence of allosteric regulators or active site ligands, by hanging drop vapour diffusion method, mixing of 0.002 ml of 9.7 mg/ml protein solution with an equal volume of reservoir solution containing 8% PEG 8000, 15% MPD, 0.4 M NaCl and 0.1 M phosphate, pH 7.0, and equilibration against 1.0 ml of reservoir solution at 18°C, method screening and optimization, X-ray diffraction structure determination and analysis at 2.9 A resolution, molecular replacement method and modelling using the hexameric human apo-enzyme hGDH1 structure (PDB ID 1L1F) as a search model

Homo sapiens

Protein Variants

Protein Variants	Comment	Organism
G456A	naturally occuring mutation, an evolutionary amino acid substitution compared to hGDH1 which confers GTP resistance, residue 456 from the pivot helix has a key role in the transition between closed and open conformations, a process which includes movements of the pivot helix and the NAD+-binding domain, leading to the opening of the active site cleft. Local flexibility is affected by an intersubunit hydrophobic interaction at the base of the antenna between residues Phe387 and Leu401. In GDH1, flexibility is also affected by the presence of the small and flexible Gly456 residue which packs against the Phe and Leu. Replacement of Gly by the bulkier and less flexible Ala456 in hGDH2 is expected to reduce local flexibility, and thus to affect the opening and closing of the active site cleft	Homo sapiens
G456A	site-directed mutagenesis, residue 456 from the pivot helix has a key role in the transition between closed and open conformations, a process which includes movements of the pivot helix and the NAD+-binding domain, leading to the opening of the active site cleft. Local flexibility is affected by an intersubunit hydrophobic interaction at the base of the antenna between residues Phe387 and Leu401. In GDH1, flexibility is also affected by the presence of the small and flexible Gly456 residue which packs against the Phe and Leu. Replacement of Gly by the bulkier and less flexible Ala456 as in hGDH2 is expected to reduce local flexibility, and thus to affect the opening and closing of the active site cleft	Homo sapiens
M415L/R443S/G456A/R470H	site-directed mutagenesis	Homo sapiens
R443S	naturally occuring mutation, an evolutionary amino acid substitution which is associated with lower basal activity, though still permitting activation by ADP. Replacement of the GDH1 residue Arg443 by Ser in GDH2 was a key event early in the evolution of the GLUD2 gene in humans and great apes, which has been related with a lower basal activity and heat sensitivity. Arg443 is involved in the stabilization of open and closed conformations of GDH1. GDH1 structures reveal that the transition from open to closed conformations is associated with partial unfolding of the C-terminus of the descending helix, immediately after residue Arg443, thereby reducing the length of the helix by almost a half turn. Arg443 forms conserved intersubunit hydrogen bonds with backbone and/or side chains of Asp408 and Ser409 residues from the ascending helix of a neighbouring chain and a conserved stacking interaction with Tyr405. Through these interactions, Arg443 establishes crucial intersubunit connections that mutually stabilize the ascending and descending helices. In contrast, substitution of Arg443 by Ser in hGDH2 leads to a loss of the above interactions, and a loose packing in the antenna region. This results in a higher flexibility in the area and in a lower stability of the enzyme, which subsequently may contribute to a lower enzymatic basal activity and an increased heat sensitivity	Homo sapiens
R443S	site-directed mutagenesis, replacement of the GDH1 residue Arg443 by Ser in GDH2 was a key event early in the evolution of the GLUD2 gene in humans and great apes, which has been related with a lower basal activity and heat sensitivity. Arg443 is involved in the stabilization of open and closed conformations of GDH1. GDH1 structures reveal that the transition from open to closed conformations is associated with partial unfolding of the C-terminus of the descending helix, immediately after residue Arg443, thereby reducing the length of the helix by almost a half turn. Arg443 forms conserved intersubunit hydrogen bonds with backbone and/or side chains of Asp408 and Ser409 residues from the ascending helix of a neighbouring chain and a conserved stacking interaction with Tyr405. Through these interactions, Arg443 establishes crucial intersubunit connections that mutually stabilize the ascending and descending helices. In contrast, substitution of Arg443 by Ser in hGDH2 leads to a loss of the above interactions, and a loose packing in the antenna region. This results in a higher flexibility in the area and in a lower stability of the enzyme, as we observed in earlier studies, which subsequently may contribute to a lower enzymatic basal activity and an increased heat sensitivity	Homo sapiens
R470H	naturally occuring mutation, an evolutionary amino acid substitution	Homo sapiens

Inhibitors

Inhibitors	Comment	Organism	Structure
additional information	the isozyme hGDH2 is resistant against GTP inhibition	Homo sapiens

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining
mitochondrion	-	Homo sapiens	5739	-

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
L-glutamate + H2O + NAD(P)+	Homo sapiens	-	2-oxoglutarate + NH3 + NAD(P)H + H+	-	r

Organism

Organism	UniProt	Comment	Textmining
Homo sapiens	P00367	-	-
Homo sapiens	P49448	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant isozyme hGDH1 mutants from Spodoptera frugiperda SF21 cells by ammonium sulfate fractionation, hydrophobic interaction chromatography, and hydroxyapatite chromatography	Homo sapiens
recombinant isozyme hGDH2 from Spodoptera frugiperda SF21 cells by ammonium sulfate fractionation, hydrophobic interaction chromatography, and hydroxyapatite chromatography	Homo sapiens

Source Tissue

Source Tissue	Comment	Organism	Textmining
astrocyte	-	Homo sapiens	-
brain	-	Homo sapiens	-
carcinoma cell	-	Homo sapiens	-
neuron	-	Homo sapiens	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
L-glutamate + H2O + NAD(P)+	-	Homo sapiens	2-oxoglutarate + NH3 + NAD(P)H + H+	-	r

Subunits

Subunits	Comment	Organism
homohexamer	-	Homo sapiens
homohexamer	dimer of trimers, the enzyme adopts a novel semi-closed conformation, which is an intermediate between known open and closed GDH1 conformations, differing from both. Structure-function analysis, overview. Each monomer consists of the glutamate-binding domain (residues 1-210), the NAD+-binding domain (residues 211-399), the antenna (residues 400-448), the pivot helix (residues 449-478) and the C-terminal alpha-helix (residues 479-501), which are very similar to the structural features of hGDH1. The antenna region is formed by the ascending and descending helix	Homo sapiens

Synonyms

Synonyms	Comment	Organism
GDH1	-	Homo sapiens
GDH2	-	Homo sapiens
GLUD1	-	Homo sapiens
GLUD2	-	Homo sapiens
glutamate dehydrogenase 1	-	Homo sapiens
glutamate dehydrogenase 2	-	Homo sapiens
hGDH1	-	Homo sapiens
hGDH2	-	Homo sapiens

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
22	-	assay at room temperature	Homo sapiens

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
8	-	assay at, reductive amination	Homo sapiens

Cofactor

Cofactor	Comment	Organism
NAD+	-	Homo sapiens
NADH	-	Homo sapiens
NADP+	-	Homo sapiens
NADPH	-	Homo sapiens

General Information

General Information	Comment	Organism
evolution	hGDH2 emerged recently via retroposition during primate evolution, being only present in humans and some closely related great apes. Functional evolution of hGDH isoenzymes, overview. Reflecting the very recent emergence of hGDH2 from hGDH1, the two human proteins show very high amino acid sequence homology (about 97%), differing in only 15 of 505 amino acids in their mature forms. Despite this similarity, hGDH2 has unique enzymatic and regulatory properties. These include GTP resistance and low basal activity amenable to activation by ADP and/or L-leucine, lower optimal pH and relative sensitivity to thermal inactivation. These properties are to a large extent associated with only two of the 15 amino acid substitutions that occurred in the course of hGDH2 evolution. In particular, the Gly456 to Ala substitution confers GTP resistance, whereas the Arg443 to Ser change is associated with lower basal activity, though still permitting activation by ADP	Homo sapiens
evolution	reflecting the very recent emergence of hGDH2 from hGDH1, the two human proteins show very high amino acid sequence homology (about 97%), differing in only 15 of 505 amino acids in their mature forms. Despite this similarity, hGDH2 has unique enzymatic and regulatory properties	Homo sapiens
additional information	structure-function analysis, overview. Structure comparison with isozyme hGDH1, comparison of open, semi-closed and closed conformations from apo-hGDH1 (PDB ID 1L1F) and hGDH2 (PDB ID 6G2U)	Homo sapiens
additional information	structure-function analysis, overview. Structure comparison with isozyme hGDH2, comparison of open, semi-closed and closed conformations from apo-hGDH1 (PDB ID 1L1F) and hGDH2 (PDB ID 6G2U)	Homo sapiens
physiological function	isozyme hGDH2 is found in both human astrocytes and neurons, where it is thought to contribute to glutamate handling, both as a neurotransmitter and as a metabolic intermediate. It plays a putative role in early nervous system development, neurodegenerative processes, and oncogenesis. Regarding its role in cancer pathophysiology, hGDH2 promotes tumor cell survival especially under deprived conditions, such as glucose or glutamine depletion	Homo sapiens