Information on EC 3.5.1.78 - Glutathionylspermidine amidase

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

EC NUMBER
COMMENTARY hide
3.5.1.78
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RECOMMENDED NAME
GeneOntology No.
Glutathionylspermidine amidase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
glutathionylspermidine + H2O = glutathione + spermidine
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of linear amides
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Glutathione metabolism
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Metabolic pathways
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SYSTEMATIC NAME
IUBMB Comments
gamma-L-Glutamyl-L-cysteinyl-glycine:spermidine amidase
Spermidine is numbered so that atom N-1 is in the amino group of the aminopropyl part of the molecule. The enzyme from Escherichia coli is bifunctional and also catalyses the glutathionylspermidine synthase (EC 6.3.1.8) reaction, resulting in a net hydrolysis of ATP.
CAS REGISTRY NUMBER
COMMENTARY hide
171040-71-4
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
bifunctional glutathionylspermidine synthetase, EC 6.3.1.8, and amidase, EC 3.5.1.78
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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
evolution
metabolism
microarray studies comparing gss+ and DELTAgss strains of Escherichia coli show that a large number of genes are either upregulated (76 genes more than 3fold) or downregulated (35 genes more than 3fold) by the loss of the gss gene. Most significant categories of up-regulated genes include sulfur utilization, glutamine and succinate metabolism, polyamine and arginine metabolism, and purine and pyrimidine metabolism
additional information
comparison of catalytic properties of recombinant purified proteins GspSA, YgiC, and YjfC, overview
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
gamma-Glu-Ala-Gly-4-nitroanilide + H2O
?
show the reaction diagram
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glutathionylspermidine + H2O
glutathione + spermidine
show the reaction diagram
glutathionylspermidine disulfide + H2O
glutathione + ?
show the reaction diagram
a coupled reaction of Gsp amidase and glutathione reductase
products of the coupled reaction of Gsp amidase and glutathione reductase: spermidine, glutathionylspermidine-glutathione mixed disulfide, and glutathione disulfide are generated
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?
trypanothione + H2O
glutathione + glutathionylspermidine
show the reaction diagram
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
glutathionylspermidine + H2O
glutathione + spermidine
show the reaction diagram
additional information
?
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P0AES0
bifunctional enzyme that catalyzes the ATP-dependent formation and hydrolysis of glutathionylspermidine, a conjugate of glutathione and spermidine. Proteins YgiC and YjfC, encoded by genes ygiC and yjfC, show ATPase activity, but are not glutathionylspermidine synthetases
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
ATP
signature amino acid residues of ATP-grasp domain, R316, K498, K533, are responsible for the ATP binding
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
gamma-Glu-Ala-Gly-CHO
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most probably captures Cys59 and accumulates as the tetrahedral adduct in the amidase active site. Binding of phosphinophosphate in the Gsp synthetase active site potentiates the inhibition affinity for the aldehyde at the Gsp amidase active site by two orders of magnitude
iodoacetamide
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L-gamma-glutamyl-5-azido-N-(3-[[(4-methylphenyl)sulfonyl]oxy]-2-oxopropyl)-L-norleucinamide
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L-gamma-glutamyl-5-azido-N-[3-(benzoyloxy)-2-oxopropyl]-L-norleucinamide
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L-gamma-glutamyl-N-[3-(benzoyloxy)-2-oxopropyl]-6-[4-([[5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoyl]amino]methyl)-1H-1,2,3-triazol-1-yl]-L-norleucinamide
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Polyamine-containing phosphopeptides
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potent and selective inhibitors, which selectively inhibit the synthetase domain over the amidase domain
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.114
ATP
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pH 7.3, synthetase activity
0.5 - 5.5
glutathionylspermidine
0.242
reduced glutathione
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pH 7.3, synthetase activity
0.059
spermidine
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pH 7.3, synthetase activity
additional information
additional information
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2.1
glutathionylspermidine
Escherichia coli
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IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00075 - 0.00217
L-gamma-glutamyl-5-azido-N-(3-[[(4-methylphenyl)sulfonyl]oxy]-2-oxopropyl)-L-norleucinamide
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0.00068
L-gamma-glutamyl-5-azido-N-[3-(benzoyloxy)-2-oxopropyl]-L-norleucinamide
Escherichia coli
P0AES0
pH and temperature not specified in the publication
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
45000
2 * 45000, SDS-PAGE, homodimer in which each monomer consists of two distinct domains. The C-terminal domain is responsible for the synthesis of glutathionylspermidine while the N-terminal domain catalyzes the hydrolysis of the conjugate
70000
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or dimer, 3 or 2 * 70000, SDS-PAGE; or trimer, 2 or 3 * 70000, SDS-PAGE
170000
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gel filtration, the deviation of the estimated molecular mass from 140000 of a dimer might result from the dimer having a non-globular shape
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
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or trimer, 2 or 3 * 70000, SDS-PAGE
homodimer
2 * 45000, SDS-PAGE, homodimer in which each monomer consists of two distinct domains. The C-terminal domain is responsible for the synthesis of glutathionylspermidine while the N-terminal domain catalyzes the hydrolysis of the conjugate
trimer
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or dimer, 3 or 2 * 70000, SDS-PAGE
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
mutant enzyme C59A in complex with glutathionylspermidine
hanging gamma-drop vapor diffusion method, structure of Leishmania major trypanothione synthetase-amidase, determined in three crystal forms, reveals two catalytic domains
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
90
purified recombinant enzyme, pH 6.8, 3 min, inactivation
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
the presence of Mg2+, ATP and reduced glutathione, with or without spermidine, markedly protects the enzyme from cleavage by trypsin
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
nickel affinity column chromatography
recombinant enzyme
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recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by anion exchange chromatography and gel filtration
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21(DE3) cells
expression in Escherichia coli, His6-tag
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gene gsp, DNA and amino acid sequence determination and analysis, sequence comparisons, recombinant expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
gene gss, phylogenetic and global transcriptome analyses, microarray studies comparing gss+ and DELTAgss strains of Escherichia coli show that a large number of genes are either upregulated (76 genes more than 3fold) or downregulated (35 genes more than 3fold) by the loss of the gss gene. Most significant categories of up-regulated genes include sulfur utilization, glutamine and succinate metabolism, polyamine and arginine metabolism, and purine and pyrimidine metabolism
mutant enzyme C59A is expressed in Escherichia coli BL21(DE3) cells
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C79A
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complete loss of amidase activity without affecting synthetase activity
additional information
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
H2O2-mediated inactivation of the amidase activity can be recovered by the addition of 4 mM glutathione. ca. 80% of the enzyme activity are rescued
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