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Information on EC 6.3.1.9 - trypanothione synthase and Organism(s) Crithidia fasciculata and UniProt Accession O60993
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EC Tree
6.3.1.9 trypanothione synthaseIUBMB Comments
The enzyme, characterized from several trypanosomatids (e.g. Trypanosoma cruzi) catalyses two subsequent reactions, leading to production of trypanothione from glutathione and spermidine. Some trypanosomatids (e.g. Crithidia species and some Leishmania species) also contain an enzyme that only carries out the first reaction (cf. EC 6.3.1.8, glutathionylspermidine synthase). The enzyme is bifunctional, and also catalyses the hydrolysis of glutathionylspermidine and trypanothione (cf. EC 3.5.1.78, glutathionylspermidine amidase).
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Word Map
- 6.3.1.9
- trypanosoma
- leishmania
-
trypanosomatids
- brucei
- cruzi
- fasciculata
- crithidia
-
n1,n8-bisglutathionylspermidine
-
chagas
- infantum
-
tryparedoxine
-
antileishmanial
-
parasite-specific
- kinetoplastida
-
antitrypanosomal
- drug development
- synthesis
- pharmacology
The taxonomic range for the selected organisms is: Crithidia fasciculata
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota
Reaction Schemes
Synonyms
trypanothione synthetase, tctrys, ldtrys, tsh synthetase, litrys, tbtrys, trypanothione synthase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
Cf-TS
-
-
-
-
GenBank AF006615-derived protein GI 3004644
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-
-
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Synthetase, trypanothione
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-
-
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Synthetase, trypanothione (Crithidia fasciculata strain HS6 gene Cf-TS)
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-
-
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Trypanothione synthetase
-
-
-
-
Trypanothione synthetase (Crithidia fasciculata strain HS6 gene Cf-TS)
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-
-
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TSH synthetase
-
-
-
-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
glutathione + glutathionylspermidine + ATP = N1,N8-bis(glutathionyl)spermidine + ADP + phosphate
the reaction is catalyzed by trypanothione synthase, which also catalyzes the reaction of glutathionyspermidine synthase, EC 6.3.1.8, in Crithidia fasciculata, trypanothione synthase further exhibits amidase and marginal ATPase activities
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Acid amide formation
-
-
-
-
SYSTEMATIC NAME
IUBMB Comments
spermidine/glutathionylspermidine:glutathione ligase (ADP-forming)
The enzyme, characterized from several trypanosomatids (e.g. Trypanosoma cruzi) catalyses two subsequent reactions, leading to production of trypanothione from glutathione and spermidine. Some trypanosomatids (e.g. Crithidia species and some Leishmania species) also contain an enzyme that only carries out the first reaction (cf. EC 6.3.1.8, glutathionylspermidine synthase). The enzyme is bifunctional, and also catalyses the hydrolysis of glutathionylspermidine and trypanothione (cf. EC 3.5.1.78, glutathionylspermidine amidase).
CAS REGISTRY NUMBER
COMMENTARY
130246-69-4
-
213260-30-1
synthetase, trypanothione (Crithidia fasciculata strain HS6 gene Cf-TS) /GenBank AF006615-derived protein GI 3004644 /trypanothione synthetase (Crithidia fasciculata strain HS6 gene Cf-TS)
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
gamma-L-Glu-L-Cys-Gly + N1-(gamma-Glu-L-Cys-Gly)-spermidine + ATP
N1,N8-bis-(gamma-L-glutamyl-L-cysteinyl-glycyl)-spermidine + ADP + phosphate
synthesis of trypanothione, which is involved in maintaining intracellular thiol redox and in defense against oxidants
-
-
?
2 glutathione + spermidine + 2 ATP
N1,N8-bis(glutathionyl)spermidine + 2 ADP + 2 phosphate
trypanothione synthase overall reaction
-
-
?
gamma-L-Glu-L-alpha-aminobutyrylglycine + N1-(gamma-Glu-L-Cys-Gly)-spermidine + ATP
?
-
gamma-L-Glu-L-alpha-aminobutyrylglycine i.e. ophthalmic acid
-
-
?
gamma-L-Glu-L-Cys-Gly + N1-(gamma-Glu-L-Cys-Gly)-spermidine + ATP
N1,N8-bis-(gamma-L-glutamyl-L-cysteinyl-glycyl)-spermidine + ADP + phosphate
-
the enzyme is involved in the biosynthesis of the trypanosomatid-specific dithiol trypanothione
-
-
?
gamma-L-Glu-L-Cys-Gly + N1-(gamma-Glu-L-Cys-Gly)-spermidine + ATP + H2O
N1,N8-bis-(gamma-L-Glu-L-Cys-Gly)-spermidine + ADP + phosphate + NH3
glutathione + glutathionylspermidine + ATP
N1,N8-bis(glutathionyl)spermidine + ADP + phosphate
glutathione + N8-acetylspermidine + ATP
N1-glutathionyl-N8-acetylspermidine + ADP + phosphate
-
-
-
?
N8-glutathionylspermidine + glutathione + ATP
N1,N8-bis(glutathionyl)spermidine + ADP + phosphate
-
-
-
-
?
gamma-L-Glu-L-Cys-Gly + N1-(gamma-Glu-L-Cys-Gly)-spermidine + ATP + H2O
N1,N8-bis-(gamma-L-Glu-L-Cys-Gly)-spermidine + ADP + phosphate + NH3
-
-
-
-
?
gamma-L-Glu-L-Cys-Gly + N1-(gamma-Glu-L-Cys-Gly)-spermidine + ATP + H2O
N1,N8-bis-(gamma-L-Glu-L-Cys-Gly)-spermidine + ADP + phosphate + NH3
-
-
N1-glutathionylspermidine and N8-glutathionylspermidine are intermediates
?
glutathione + glutathionylspermidine + ATP
N1,N8-bis(glutathionyl)spermidine + ADP + phosphate
-
-
-
?
glutathione + glutathionylspermidine + ATP
N1,N8-bis(glutathionyl)spermidine + ADP + phosphate
dynamic interactions of remote domains in substrate binding and catalysis
-
-
?
glutathione + spermidine + ATP
glutathionylspermidine + ADP + phosphate
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ATP in form of MgATP2-
-
-
?
glutathione + spermidine + ATP
glutathionylspermidine + ADP + phosphate
ATP in form of MgATP2-
-
-
?
additional information
?
-
the trypanothione synthase also catalyzes the reaction of glutathionylspermidine synthase, EC 6.3.1.8
-
-
?
additional information
?
-
-
the trypanothione synthase also catalyzes the reaction of glutathionylspermidine synthase, EC 6.3.1.8
-
-
?
additional information
?
-
the trypanothione synthase also catalyzes the reaction of glutathionylspermidine synthase, EC 6.3.1.8, and exhibits amidase and marginal ATPase activities
-
-
?
additional information
?
-
-
the trypanothione synthase also catalyzes the reaction of glutathionylspermidine synthase, EC 6.3.1.8, and exhibits amidase and marginal ATPase activities
-
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
gamma-L-Glu-L-Cys-Gly + N1-(gamma-Glu-L-Cys-Gly)-spermidine + ATP
N1,N8-bis-(gamma-L-glutamyl-L-cysteinyl-glycyl)-spermidine + ADP + phosphate
synthesis of trypanothione, which is involved in maintaining intracellular thiol redox and in defense against oxidants
-
-
?
gamma-L-Glu-L-Cys-Gly + N1-(gamma-Glu-L-Cys-Gly)-spermidine + ATP
N1,N8-bis-(gamma-L-glutamyl-L-cysteinyl-glycyl)-spermidine + ADP + phosphate
-
the enzyme is involved in the biosynthesis of the trypanosomatid-specific dithiol trypanothione
-
-
?
glutathione + glutathionylspermidine + ATP
N1,N8-bis(glutathionyl)spermidine + ADP + phosphate
-
-
-
?
additional information
?
-
the trypanothione synthase also catalyzes the reaction of glutathionylspermidine synthase, EC 6.3.1.8
-
-
?
additional information
?
-
-
the trypanothione synthase also catalyzes the reaction of glutathionylspermidine synthase, EC 6.3.1.8
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Mg2+
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
limited proteolysis with trypsin or factor Xa result in cleavage of wild-type and mutant enzymes at R553 followed by loss of activity, cleavage of wild-type and mutant R553Q can be delayed by presence of substrates
-
additional information
-
limited proteolysis with trypsin or factor Xa result in cleavage of wild-type and mutant enzymes at R553 followed by loss of activity, cleavage of wild-type and mutant R553Q can be delayed by presence of substrates
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.407
glutathione
pH 7.2, 25°C, recombinant trypanothione synthase
0.48
glutathionylspermidine
pH 7.2, 25°C, recombinant trypanothione synthase
additional information
additional information
steady-state kinetics for the multiple enzyme activities, Dalziel equation, overview
-
additional information
additional information
-
steady-state kinetics for the multiple enzyme activities, Dalziel equation, overview
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
8.7
glutathionylspermidine
pH 7.2, 25°C, recombinant trypanothione synthase
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
2.1 - 2.2
purified recombinant trypanothione synthase performing the trypanothione synthase reaction, average of the activity of recombinant enzymes expressed from different constructs, overview
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7.5 - 7.8
-
synthesis of both mono-glutathionylspermidine and di-glutathionylspermidine conjugates
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). TRYS_CRIFA
652
0
74517
Swiss-Prot
other Location (Reliability: 4)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
-
in vivo a complex between EC 6.3.1.8 and EC 6.3.1.9 exists that persists during purification
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
C59A
-
mutant lacks the amidase activity, while the synthetase activity is similar to wild-type. Constructed for the production of trypanothione and trypanothione disulfide in >200 mg quantities. The protocol also allows the synthesis of related glutathione conjugates
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant His-tagged wild-type enzyme and mutants from Escherichia coli by nickel affinity chromatography
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
DNA sequence determination and analysis, expression of wild-type enzyme and mutants in Escherichia coli as His-tagged or Nus-fusion proteins
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pharmacology
-
the enzyme would serve as a potential target for antiparasitic chemotherapy
synthesis
-
production of trypanothione and trypanothione disulfide in >200 mg quantities by mutant C59A lacking the amidase activity. The protocol also allows the synthesis of related glutathione conjugates
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Nadeau, K.C.
Biochemical studies on protein folding chaperones (HSP90 and cyclophilin) and on trypanosomal enzymes (trypanothione and glutathionylspermidine synthetases) (heat shock protein)
Diss. Abstr. Int. B
56
3744
1995
Crithidia fasciculata
-
Smith, K.; Nadeau, K.; Bradley, M.; Walsh, C.; Fairlamb, A.H.
Purification of glutathionylspermidine and trypanothione synthetase from Crithidia fasciculata
Protein Sci.
1
874-883
1992
Crithidia fasciculata
Tetaud, E.; Manai, F.; Barrett, M.P.; Nadeau, K.; Walsh, C.T.; Fairlamb, A.H.
Cloning and characterization of the two enzymes responsible for trypanothione biosynthesis in Crithidia fasciculata
J. Biol. Chem.
273
19383-19390
1998
Crithidia fasciculata (O60993), Crithidia fasciculata
Henderson, G.B.; Yamaguchi, M.; Novoa, L.; Fairlamb, A.H.; Cerami, A.
Biosynthesis of the trypanosomatid metabolite trypanothione: purification and characterization of trypanothione synthetase from Crithidia fasciculata
Biochemistry
29
3924-3929
1990
Crithidia fasciculata
Comini, M.; Menge, U.; Wissing, J.; Flohe, L.
Trypanothione synthesis in Crithidia revisited
J. Biol. Chem.
280
6850-6860
2005
Crithidia fasciculata (Q5DM89), Crithidia fasciculata
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