Information on EC 6.3.1.8 - Glutathionylspermidine synthase

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

EC NUMBER
COMMENTARY hide
6.3.1.8
-
RECOMMENDED NAME
GeneOntology No.
Glutathionylspermidine synthase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
glutathione + spermidine + ATP = glutathionylspermidine + ADP + phosphate
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Acid amide formation
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
glutathione metabolism
-
-
Glutathione metabolism
-
-
glutathionylspermidine biosynthesis
-
-
Metabolic pathways
-
-
SYSTEMATIC NAME
IUBMB Comments
gamma-L-Glutamyl-L-cysteinyl-glycine:spermidine ligase (ADP-forming) [spermidine is numbered so that atom N-1 is in the amino group of the aminopropyl part of the molecule]
Requires magnesium ions. Involved in the synthesis of trypanothione in trypanosomatids. The enzyme from Escherichia coli is bifunctional and also catalyses the glutathionylspermidine amidase (EC 3.5.1.78) reaction, resulting in a net hydrolysis of ATP.
CAS REGISTRY NUMBER
COMMENTARY hide
168257-36-1
synthetase, glutathionylspermidine (Escherichia coli clone pJBM1 gene gsp), GenBank AE000381-derived protein GI 1789361, protein (Escherichia coli strain K12-MG1655 gene gsp)
190209-90-6
synthetase, glutathionylspermidine (Crithidia fasciculata), glutathionylspermidine synthetase (Crithidia fasciculata)
209273-40-5
synthetase, glutathionylspermidine (Crithidia fasciculata fragment)
213260-31-2
synthetase, glutathionylspermidine (Crithidia fasciculata strain HS6 gene Cf-GSS), genBank U66520-derived protein GI 1813514, glutathionylspermidine synthetase (Crithidia fasciculata strain HS6 gene Cf-GSS)
907709-2
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
gene gss
UniProt
Manually annotated by BRENDA team
inactive gene GSPS; inactive gene GSPS
AJ748279
GenBank
Manually annotated by BRENDA team
bifunctional enzyme with glutathionylspermidine synthase and trypanothione synthase activity
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
-
hypersensitivities of the GspSA/glutaredoxin null mutants to H2O2 support the idea that GspSA and Grx synergistically defend against oxidative damage
additional information
transcriptome analyses, overview. A large number of genes are either upregulated (76 genes more than threefold) or downregulated (35 genes more than threefold) by the loss of the gss gene
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2 glutathione + spermidine + 2 ATP
N1,N8-bis(glutathionyl)spermidine + 2 ADP + 2 phosphate
show the reaction diagram
trypanothione synthase overall reaction
-
-
?
gamma-Glu-Gly-Gly + spermidine + ATP
N1-(gamma-Glu-Gly-Gly)-spermidine + ADP + phosphate
show the reaction diagram
-
at 14% of the activity with gamma-L-Glu-L-Cys-Gly
-
-
-
gamma-Glu-L-Cys(S-butyl)-Gly + spermidine + ATP
N1-(gamma-Glu-L-Cys(S-butyl)-Gly)-spermidine + ADP + phosphate
show the reaction diagram
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at 24% of the activity with gamma-L-Glu-L-Cys-Gly
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-
-
gamma-Glu-L-Cys(S-ethyl)-Gly + spermidine + ATP
N1-(gamma-Glu-L-Cys(S-ethyl)-Gly)-spermidine + ADP + phosphate
show the reaction diagram
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at 77% of the activity with gamma-L-Glu-L-Cys-Gly
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-
-
gamma-Glu-L-Cys(S-propyl)-Gly + spermidine + ATP
N1-(gamma-Glu-L-Cys(S-propyl)-Gly)-spermidine + ADP + phosphate
show the reaction diagram
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at 56% of the activity with gamma-L-Glu-L-Cys-Gly
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-
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gamma-Glu-L-Ser-Gly + spermidine + ATP
N1-(gamma-Glu-L-Ser-Gly)-spermidine + ADP + phosphate
show the reaction diagram
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at 16% of the activity with gamma-L-Glu-L-Cys-Gly
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-
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gamma-L-Glu-L-Ala-Gly + spermidine + ATP
N1-(gamma-L-Glu-L-Ala-Gly)-spermidine + ADP + phosphate
show the reaction diagram
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at 28% of the activity with gamma-L-Glu-L-Cys-Gly
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-
-
gamma-L-Glu-L-alpha-aminobutyryl-Gly + spermidine + ATP
N1-(gamma-L-Glu-L-alpha-aminobutyryl-Gly)-spermidine
show the reaction diagram
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gamma-L-Glu-L-alpha-aminobutyryl-Gly i.e. ophthalmic acid
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-
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gamma-L-Glu-L-Cys(S-methyl)-Gly + spermidine + ATP
N1-(gamma-L-Glu-L-Cys(S-methyl)-Gly)-spermidine + ADP + phosphate
show the reaction diagram
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at 74% of the activity with gamma-L-Glu-L-Cys-Gly
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-
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gamma-L-Glu-L-Cys-Gly + spermidine + ATP
N1-(gamma-L-Glu-L-Cys-Gly)-spermidine + ADP + phosphate
show the reaction diagram
gamma-L-Glu-L-Cys-Gly + spermidine + MgATP2-
N1-(gamma-L-Glu-L-Cys-Gly)-spermidine + MgADP- + phosphate
show the reaction diagram
gamma-L-Glu-L-Cys-Gly + spermidine + MgATP2-
N1-(gamma-L-Glu-L-Cys-Gly)-spermidine + MgADP2- + phosphate
show the reaction diagram
-
substitution of the glycine part abolishes activity
-
?
gamma-L-Glu-L-Ile-Gly + spermidine + ATP
N1-(gamma-L-Glu-L-Ile-Gly)-spermidine + ADP + phosphate
show the reaction diagram
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at 70% of the activity with gamma-L-Glu-L-Cys-Gly
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-
-
gamma-L-Glu-L-Val-Gly + spermidine + ATP
N1-(gamma-L-Glu-L-Val-Gly)-spermidine + ADP + phosphate
show the reaction diagram
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at 88% of the activity with gamma-L-Glu-L-Cys-Gly
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-
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gamma-L-Glutamyl-L-cysteinyl-glycine + spermidine + ATP
?
show the reaction diagram
glutathione + spermidine + ATP
glutathionylspermidine + ADP + phosphate
show the reaction diagram
glutathionylspermidine + H2O
glutathione + spermidine
show the reaction diagram
amidase activity
-
?
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
gamma-L-Glutamyl-L-cysteinyl-glycine + spermidine + ATP
?
show the reaction diagram
glutathione + spermidine + ATP
glutathionylspermidine + ADP + phosphate
show the reaction diagram
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(2-[[([2-[(4-amino-4-carboxybutanoyl)amino]propanoyl]amino)methyl](hydroxy)phosphoryl]ethyl)(hydroxy)dioxophosphate(1-)
inhibits the reaction in the presence of spermidine
4-[(14-ammonio-5-hydroxy-1-methyl-5-oxido-2-oxo-6-oxa-3,10-diaza-5-phosphatetradec-1-yl)amino]-1-carboxy-4-oxobutan-1-aminium
-
4-[[2-([[[4-[(4-ammoniobutyl)amino]butyl](hydroxy)phosphoryl]methyl]amino)-1-methyl-2-oxoethyl]amino]-1-carboxy-4-oxobutan-1-aminium
-
buthionine sulfoximine
-
-
gamma-Aminobutyryl-L-Ala-Gly
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5 mM, 10% inhibition
gamma-glutamyl-N-[[(3-aminopropoxy)(2-phosphonoethyl)phosphoryl]methyl]alaninamide
-
gamma-L-Glu-D-Phe-Gly
-
5 mM, 10% inhibition
gamma-L-Glu-L-Leu
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5 mM, 10% inhibition
gamma-L-Glu-L-Leu-Gly
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5 mM, 58% inhibition
gamma-L-Glu-L-leu-L-Ala
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5 mM, 95% inhibition
gamma-L-Glu-L-Phe-Gly
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5 mM, 73% inhibition
gamma-L-Glu-L-Ser-Gly
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5 mM, 50% inhibition
gamma-L-Glu-L-Val
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5 mM, 10% inhibition
gamma-L-Glu-L-Val-L-Ala
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5 mM, 58% inhibition
gamma-L-glutamyl-L-cysteinyl-diaminopropionic acid
-
;
Glutaryl-L-Val-Gly
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5 mM, 10% inhibition
Hydrogen O-[3-[N-(4-Aminobutyl)amino]propyl][[(gamma-glutamylalanyl)amino]methyl]phosphonate
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L-beta-Asp-L-Leu-Gly
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5 mM, 42% inhibition
L-beta-Asp-L-Val-Gly
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5 mM, 15% inhibition
L-Cys-Gly
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5 mM, 20% inhibition
Melarsen oxide
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-
N1-glutathionylspermidine analogs
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selective inhibitors against EC 6.3.1.8 and EC 3.5.1.78 activities provide evidence of interdomain communication in the bifunctional enzyme
nifurtimox
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Non-polyamine-containing phosphoamidate
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Pentamidine
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Phosphinate
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behaves as a slow-binding bisubstrate inhibitor, competitive with respect to GSH and spermidine
phosphinate analogue of glutathionylspermidine
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slow tight-binding inhibition
phosphonate
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acts as a a simple ground state analogue of glutathione
phosphonate analogue of glutathionylspermidine
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classical, linear competitive inhibition with respect to GSH
Polyamine-containing phosphapeptides
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potent and selective
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reduced trypanothione
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triostam
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additional information
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.052 - 0.215
ATP
14
gamma-Glu-L-Cys(S-butyl)-Gly
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-
0.18
gamma-Glu-L-Cys(S-ethyl)-Gly
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-
0.53
gamma-Glu-L-Cys(S-propyl)-Gly
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4
gamma-L-Glu-L-Ala-Gly
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-
0.13
gamma-L-Glu-L-Cys(S-methyl)-Gly
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-
0.91
gamma-L-Glu-L-Cys-Gly
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-
0.4
gamma-L-Glu-L-Ile-Gly
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-
0.67
gamma-L-Glu-L-Val-Gly
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-
0.0224 - 7.298
glutathione
0.5
glutathionylspermidine
-
pH 7.3, 25C, amidase activity
0.096 - 0.114
MgADP2-
0.059 - 7.424
spermidine
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
9.2
gamma-Glu-L-Cys(S-butyl)-Gly
Crithidia fasciculata
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-
11.3
gamma-Glu-L-Cys(S-ethyl)-Gly
Crithidia fasciculata
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-
8.7
gamma-Glu-L-Cys(S-propyl)-Gly
Crithidia fasciculata
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-
2.5
gamma-L-Glu-L-Ala-Gly
Crithidia fasciculata
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-
8.8
gamma-L-Glu-L-Cys(S-methyl)-Gly
Crithidia fasciculata
-
-
15
gamma-L-Glu-L-Cys-Gly
Crithidia fasciculata
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-
10
gamma-L-Glu-L-Ile-Gly
Crithidia fasciculata
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-
11.8
gamma-L-Glu-L-Val-Gly
Crithidia fasciculata
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-
4.2
spermidine
Crithidia fasciculata
Q5DM89
pH 7.2, 25C, recombinant trypanothione synthase performing the glutathionylspermidine synthase reaction
additional information
additional information
Crithidia fasciculata
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-
-
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.006
4-[(14-ammonio-5-hydroxy-1-methyl-5-oxido-2-oxo-6-oxa-3,10-diaza-5-phosphatetradec-1-yl)amino]-1-carboxy-4-oxobutan-1-aminium
-
0.0000078
4-[[2-([[[4-[(4-ammoniobutyl)amino]butyl](hydroxy)phosphoryl]methyl]amino)-1-methyl-2-oxoethyl]amino]-1-carboxy-4-oxobutan-1-aminium
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5
gamma-L-Glu-L-Ser-Gly
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-
7.2
gamma-L-glutamyl-L-cysteinyl-diaminopropionic acid
-
-
0.029
Phosphinate
-
pH 7.2, 25C
0.019
phosphinate analogue of glutathionylspermidine
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pH 7.2, 25C
0.156
phosphonate analogue of glutathionylspermidine
-
pH 7.2, 25C
additional information
additional information
-
inhibition kinetics and mechanism, model of ter-reactant mechanism of GspS catalysis and postulated, overview
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IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.072
Phosphinate
Crithidia fasciculata
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pH 7.2, 25C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
2
about, purified recombinant trypanothione synthase performing the glutathionylspermidine synthase reaction
7.9
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pH 7.3, 25C, synthetase activity
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.6 - 4.7
-
isoelectric focusing
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
80340
-
predicted from sequence
80520
-
MALDI-TOF MS
80640
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MALDI-TOF MS
85200
-
gel filtration
138000
analytical ultracentrifugation
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
-
x * 90000, SDS-PAGE
dimer
2 * 70000, analytical ultracentrifugation
monomer
additional information
-
in vivo a complex between EC 6.3.1.8 and EC 6.3.1.9 exists that persists during purification
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
hanging drop vapour diffusion method generating crystals of GspS in 0.1M TrisHCl (pH 8.5) containing 12% (v/v) PEG3350 and 0.5 M MgCl2
mutant C59A lacking amidase activity, in complex with amidase substrate gluthionylspermidine and ADP. Homodimer, and each monomer contains the N-terminal amidase and C-terminal synthetase domains connected by a linker in between
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
90
purified recombinant GspSA C59A, 3 min, inactivation
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-80C, 4 months, loses less than 10% synthetase activity
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
nickel affinity column chromatography
recombinant enzyme mutant GspSA C59A from Escherichia coli strain BL21(DE3)
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) to over 98% purity
-
using Ni-chelating and anion-exchange chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
DNA sequence determination, cloning and RNAi transfection, expression in Escherichia coli
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enzyme expression analysis, cloning and transfection of RNAi
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expressed in Escherichia coli BL21 (DE3) cells
expression in Escherichia coli and Saccharomyces cerevisiae; overexpression in Escherichia coli and Saccharomyces cerevisiae of the gene Cf-GSS yields insoluble proteins
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expression of a recombinant histidine-tagged enzyme in Escherichia coli
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expression of enzyme mutant GspSA C59A in Escherichia coli strain BL21(DE3)
gene GSPS, a pseudogene with 2 frame shift mutations and 2 stop codons, inactive enzyme, phylogenetic analysis
AJ748279
gene gspS, DNA and amino acid sequence determination and analysis, expression as His-tagged enzyme in Escherichia coli strain BL21(DE3)
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gene gss, DNA and amiino acid sequence determination and analysis, sequence comparisons and phylogenetic analysis, transcriptome analyses
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C79A
-
mutant retains synthetase activity, but loses amidase activity
C338A
increased Km compared to the wild type enzyme
E391A
increased Km compared to the wild type enzyme
E392A
increased Km compared to the wild type enzyme
K607A
increased Km compared to the wild type enzyme
R316E
no activity
R538A
increased Km compared to the wild type enzyme
R598A
decreased Km compared to the wild type enzyme
S335A
increased Km compared to the wild type enzyme
S337A
increased Km compared to the wild type enzyme
T441A
increased Km compared to the wild type enzyme
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
drug development
-
the enzyme is a target for drug development
pharmacology
-
the enzyme would serve as a potential target for antiparasitic chemotherapy
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