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Information on EC 6.3.2.17 - tetrahydrofolate synthase and Organism(s) Escherichia coli and UniProt Accession P08192

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IUBMB Comments
In some bacteria, a single protein catalyses both this activity and that of EC 6.3.2.12, dihydrofolate synthase , the combined activity of which leads to the formation of the coenzyme polyglutamated tetrahydropteroate (H4PteGlun), i.e. various tetrahydrofolates (H4folate). In contrast, the activities are located on separate proteins in most eukaryotes studied to date . In Arabidopsis thaliana, this enzyme is present as distinct isoforms in the mitochondria, the cytosol and the chloroplast. Each isoform is encoded by a separate gene, a situation that is unique among eukaryotes . As the affinity of folate-dependent enzymes increases markedly with the number of glutamic residues, the tetrahydropteroyl polyglutamates are the preferred coenzymes of C1 metabolism. (reviewed in ). The enzymes from different sources (particularly eukaryotes versus prokaryotes) have different substrate specificities with regard to one-carbon substituents and the number of glutamate residues present on the tetrahydrofolates.
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Escherichia coli
UNIPROT: P08192
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The taxonomic range for the selected organisms is: Escherichia coli
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
Synonyms
folylpolyglutamate synthetase, mthfd1l, folylpoly-gamma-glutamate synthetase, folylpolyglutamate synthase, folypolyglutamate synthetase, fpgs1, atdfb, cfpgs, mfpgs, folylpolyglutamyl synthetase, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dihydrofolate synthase/folylpolyglutamate synthase
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Folylpoly-gamma-glutamate synthetase
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Folate polyglutamate synthetase
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-
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Folylpoly(.gamma.-glutamate) synthase
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Folylpoly-.gamma.-glutamate synthase
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-
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Folylpoly-gamma-glutamate synthetase
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-
-
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Folylpoly-gamma-glutamate synthetase-dihydrofolate synthetase
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Folylpolyglutamate synthase
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-
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Folylpolyglutamate synthetase
Folylpolyglutamyl synthetase
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-
-
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Formyltetrahydropteroyldiglutamate synthetase
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N10-Formyltetrahydropteroyldiglutamate synthetase
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Synthetase, folylpolyglutamate
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Tail length regulator
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tetrahydrofolate:L-glutamate gamma-ligase (ADP-forming)
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tetrahydrofolylpolyglutamate synthase
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additional information
cf. EC 6.3.2.12
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
carboxylic acid amide formation
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-
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carboxamide formation
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-
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PATHWAY SOURCE
PATHWAYS
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SYSTEMATIC NAME
IUBMB Comments
tetrahydropteroyl-gamma-polyglutamate:L-glutamate gamma-ligase (ADP-forming)
In some bacteria, a single protein catalyses both this activity and that of EC 6.3.2.12, dihydrofolate synthase [3], the combined activity of which leads to the formation of the coenzyme polyglutamated tetrahydropteroate (H4PteGlun), i.e. various tetrahydrofolates (H4folate). In contrast, the activities are located on separate proteins in most eukaryotes studied to date [4]. In Arabidopsis thaliana, this enzyme is present as distinct isoforms in the mitochondria, the cytosol and the chloroplast. Each isoform is encoded by a separate gene, a situation that is unique among eukaryotes [4]. As the affinity of folate-dependent enzymes increases markedly with the number of glutamic residues, the tetrahydropteroyl polyglutamates are the preferred coenzymes of C1 metabolism. (reviewed in [5]). The enzymes from different sources (particularly eukaryotes versus prokaryotes) have different substrate specificities with regard to one-carbon substituents and the number of glutamate residues present on the tetrahydrofolates.
CAS REGISTRY NUMBER
COMMENTARY hide
63363-84-8
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SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ATP + 5,10-methylene-5,6,7,8-tetrahydrofolic acid + L-Glu
ADP + phosphate + 5,10-methylene-5,6,7,8-tetrahydrofolyl-L-Glu
show the reaction diagram
-
-
-
?
ATP + 5,6,7,8-tetrahydrofolic acid + L-Glu
ADP + phosphate + 5,6,7,8-tetrahydrofolyl-Glu
show the reaction diagram
-
-
-
?
ATP + 5,6,7,8-tetrahydrofolyl-gamma-(L-Glu)2 + L-Glu
ADP + phosphate + 5,6,7,8-tetrahydrofolyl-gamma-(L-Glu)3
show the reaction diagram
-
-
-
?
ATP + 7,8-dihydropteroate + L-Glu
ADP + phosphate + 7,8-dihydropteroyl-L-Glu
show the reaction diagram
-
-
-
?
ATP + tetrahydropteroyl-[gamma-Glu]n + L-glutamate
ADP + phosphate + tetrahydropteroyl-[gamma-Glu]n+1
show the reaction diagram
-
-
-
?
ATP + (6R,S)-5,6,7,8-tetrahydrofolic acid + L-glutamate
ADP + phosphate + (6S)-5,6,7,8-tetrahydrofolyl-gamma-Glu
show the reaction diagram
-
-
-
-
ir
ATP + 10-formyl-5,6,7,8-tetrahydropteroyl-Glu + Glu
ADP + phosphate + 10-formyl-5,6,7,8-tetrahydropteroyl-Glu2
show the reaction diagram
-
-
-
-
?
ATP + 5,10-methylene-5,6,7,8-tetrahydrofolate-Glu2 + L-glutamate
ADP + phosphate + 5,10-methylene-5,6,7,8-tetrahydrofolyl-Glu3
show the reaction diagram
-
-
-
?
ATP + 5,6,7,8-tetrahydrofolate-Glu2 + L-glutamate
ADP + phosphate + 5,6,7,8-tetrahydrofolyl-Glu3
show the reaction diagram
-
-
-
?
ATP + dihydropteroic acid + L-glutamate
ADP + dihydropteroyl-Glu
show the reaction diagram
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enhancement of ATP binding is observed in the presence of this substrate. The substrate causes a conformational change in the inactive fraction of the enzyme which allows it to bind ATP
-
?
additional information
?
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NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
ATP + tetrahydropteroyl-[gamma-Glu]n + L-glutamate
ADP + phosphate + tetrahydropteroyl-[gamma-Glu]n+1
show the reaction diagram
-
-
-
?
additional information
?
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the enzyme catalyzes the MgATP-dependent ligation of L-glutamate to tetrahydrofolate coenzymes or to antifolates at the gamma-carboxyl of the terminal glutamate to form polyglutamate derivatives
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?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Mg2+
-
required
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
5,6,7,8-tetrahydrofolyl-Glu2
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60% inhibition of ATP binding at 0.1 mM
dihydrofolate
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treatment of cells with trimethoprim leads to inhibition due to accumulation of dihydrofolate through the inhibition of dihydrofolate reductase. Therefore falling dihydrofolate reductase activity leads to falling folylpolyglutamate synthase activity in a domino-like cascade
Dihydropteroic acid
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5% inhibition of ATP binding at 0.1 mM
Endogenous inhibitor from Neurospora crassa
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the inhibitor is present in either polyglutamate-deficient mutants and in wild type. This factor is non-dialyzable, thermolabile and inactivated by urea and trypsin treatment
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trimethoprim
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treatment of cells leads to inhibition due to accumulation of dihydrofolate through the inhibition of dihydrofolate reductase. Therefore falling dihydrofolate reductase activity leads to falling folylpolyglutamate synthase activity in a domino-like cascade
additional information
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in Escherichia coli, the addition of L-glutamate to dihydropteroate (dihydrofolate synthetase activity) and the subsequent additions of L-glutamate to tetrahydrofolate (folylpolyglutamate synthetase (FPGS) activity) are catalyzed by the same enzyme, FolC. The presence of a folate binding site in Escherichia coli FolC, which is different from the one seen in folylpolyglutamate synthetases, provides avenues for the design of specific inhibitors of this enzyme in antimicrobial therapy
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.05 - 0.59
5,10-methylene-5,6,7,8-tetrahydrofolic acid
0.01 - 0.448
5,6,7,8-tetrahydrofolic acid
0.3
5,6,7,8-tetrahydrofolyl-gamma-(L-Glu)2
mutant D154A, pH 9.8
0.0063 - 0.98
7,8-dihydropteroate
0.037 - 5.7
ATP
0.12 - 3.3
L-Glu
0.014
10-formyl-5,6,7,8-tetrahydropteroyl-Glu
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-
0.0665 - 0.07
ATP
0.333
Glu
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reaction with 10-formyl-5,6,7,8-tetrahydropteroyl-Glu
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
additional information
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.138
chimera containing N-terminal residues 1-81 of Escherichia coli enzyme, residues 72-82 of Lactobacillus casei enzyme, and residues 93-422 of Escherichia coli enzyme, substrate 5,10-methylene-5,6,7,8-tetrahydrofolic acid, pH 9.8
0.376
chimera containing N-terminal residues 1-81 of Escherichia coli enzyme, residues 72-82 of Lactobacillus casei enzyme, and residues 93-422 of Escherichia coli enzyme, substrate 7,8-dihydropteroate, pH 9.8
0.912
chimera containing N-terminal residues 1-71 of Lactobacillus casei enzyme, residues 82-93 of Escherichia coli enzyme, and residues 83-428 of Lactobacillus casei enzyme, substrate 5,10-methylene-5,6,7,8-tetrahydrofolic acid, pH 9.8
1.3
chimera containing N-terminal residues 1-298 of Lactobacillus casei enzyme, residues 288-422 of Escherichia coli enzyme, substrate 5,10-methylene-5,6,7,8-tetrahydrofolic acid, pH 9.8
13.2
substrate 5,10-methylene-5,6,7,8-tetrahydrofolic acid, pH 9.8
4.1
substrate 7,8-dihydropteroate, pH 9.8
0.2
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wild type enzyme, pH 9.75
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
46000
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gel filtration
47000
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1 * 47000, SDS-PAGE
48000
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x * 48000, SDS-PAGE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 48000, SDS-PAGE
monomer
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
hanging drop method in 1.2-1.7 M ammonium sulfate
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A155H
drastic increases in Km values
A155W
13fold increase in Km value for dihydropteroate
D154A
mutation in residue specific for dihydropteroate binding, 200fold increase in Km value for substrate 5,6,7,8-tetrahydropteroyl-gamma-(L-Glu)2
T122H
drastic increases in Km values
T122W
drastic increases in Km values
E146Q
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completely inactive, no ATP binding is observed with this mutant
additional information
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
DE-52 cellulose column chromatography
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli strain BL21
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high expression
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the folC gene from Escherichia coli is cloned into plasmid pET29. The resulting plasmid, pVRC1432, is transferred into the Escherichia coli BL21 strain
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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Bognar, A.L.; Shane, B.
Bacterial folylpoly(gamma-glutamate) synthase-dihydrofolate synthase
Methods Enzymol.
122
349-359
1986
Corynebacterium sp., Escherichia coli, Lacticaseibacillus casei
Manually annotated by BRENDA team
Bognar, A.L.; Osborne, C.; Shane, B.; Singer, S.C.; Ferone, R.
Folylpoly-gamma-glutamate synthetase-dihydrofolate synthetase. Cloning and high expression of the Escherichia coli folC gene and purification and properties of the gene product
J. Biol. Chem.
260
5625-5630
1985
Escherichia coli
Manually annotated by BRENDA team
Shane, B.
Folylpoly-gamma-glutamate synthetase
Chem. Biol. Pteridines, Proc. Int. Symp. Pteridines Folic Acid Deriv. Chem. Biol. Clin. Aspects
719-728
1986
Corynebacterium sp., Escherichia coli, Lacticaseibacillus casei, Sus scrofa
-
Manually annotated by BRENDA team
Cichowicz, D.J.; Foo, S.K.; Shane, B.
Folylpoly-gamma-glutamate synthesis by bacteria and mammalian cells
Mol. Cell. Biochem.
39
209-228
1981
Cricetulus griseus, Corynebacterium sp., Escherichia coli, Enterococcus faecalis, Lacticaseibacillus casei, Neurospora crassa, Rattus norvegicus, Sus scrofa
Manually annotated by BRENDA team
Pyne, C.; Bognar, A.L.
Replacement of the folC gene, encoding folylpolyglutamate synthetase-dihydrofolate synthetase in Escherichia coli, with genes mutagenized in vitro
J. Bacteriol.
174
1750-1759
1992
Escherichia coli
Manually annotated by BRENDA team
Kimlova, L.J.; Pyne, C.; Keshavjee, K.; Huy, J.; Beebakhee, G.; Bognar, A.L.
Mutagenesis of the folC gene encoding folylpolyglutamate synthetase-dihydrofolate synthetase in Escherichia coli
Arch. Biochem. Biophys.
284
9-16
1991
Escherichia coli
Manually annotated by BRENDA team
Shane, B.; Garrow, T.; Brenner, A.; Chen, L.; Choi, Y.J.; Hsu, J.C.; Stover, P.
Folylpoly-gamma-glutamate synthetase
Chem. Biol. Pteridines Folates (J. E. Ayling et al eds. ) Plenum Press New York
629-634
1993
Saccharomyces cerevisiae, Corynebacterium sp., Escherichia coli, eukaryota, Homo sapiens, Lacticaseibacillus casei, Sus scrofa
-
Manually annotated by BRENDA team
Cossins, E.A.; Chan, P.Y.
An endogenous inhibitor of folylpolyglutamate synthetase in Neurospora crassa
Phytochemistry
27
3391-3399
1988
Bos taurus, Escherichia coli, Neurospora crassa, Pisum sativum
-
Manually annotated by BRENDA team
Sheng, Y.; Cross, J.A.; Shen, Y.; Smith, C.A.; Bognar, A.L.
Mutation of an essential glutamate residue in folylpolyglutamate synthetase and activation of the enzyme by pteroate binding
Arch. Biochem. Biophys.
402
94-103
2002
Escherichia coli, Lacticaseibacillus casei
Manually annotated by BRENDA team
Mathieu, M.; Debousker, G.; Vincent, S.; Viviani, F.; Bamas-Jacques, N.; Mikol, V.
Escherichia coli FolC structure reveals an unexpected dihydrofolate binding site providing an attractive target for anti-microbial therapy
J. Biol. Chem.
280
18916-18922
2005
Escherichia coli
Manually annotated by BRENDA team
Naponelli, V.; Hanson, A.D.; Gregory, J.F.
Improved methods for the preparation of [(3)H]folate polyglutamates: biosynthesis with Lactobacillus casei and enzymatic synthesis with Escherichia coli folylpolyglutamate synthetase
Anal. Biochem.
371
127-134
2007
Escherichia coli
Manually annotated by BRENDA team
Sheng, Y.; Khanam, N.; Tsaksis, Y.; Shi, X.M.; Lu, Q.S.; Bognar, A.L.
Mutagenesis of folylpolyglutamate synthetase indicates that dihydropteroate and tetrahydrofolate bind to the same site
Biochemistry
47
2388-2396
2008
Escherichia coli (P08192), Escherichia coli, Lacticaseibacillus casei (P15925), Lacticaseibacillus casei
Manually annotated by BRENDA team
Kwon, Y.K.; Lu, W.; Melamud, E.; Khanam, N.; Bognar, A.; Rabinowitz, J.D.
A domino effect in antifolate drug action in Escherichia coli
Nat. Chem. Biol.
4
602-608
2008
Escherichia coli
Manually annotated by BRENDA team
Raz, S.; Stark, M.; Assaraf, Y.G.
Folylpoly-gamma-glutamate synthetase A key determinant of folate homeostasis and antifolate resistance in cancer
Drug Resist. Updat.
28
43-64
2016
Rattus norvegicus (M0R401), Escherichia coli (P08192), Lacticaseibacillus casei (P15925), Mus musculus (P48760), Homo sapiens (Q05932), Homo sapiens (Q96LE3), Cricetulus griseus (Q924L9)
Manually annotated by BRENDA team