Information on EC 6.3.2.12 - dihydrofolate synthase

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

EC NUMBER
COMMENTARY hide
6.3.2.12
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RECOMMENDED NAME
GeneOntology No.
dihydrofolate synthase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + 7,8-dihydropteroate + L-glutamate = ADP + phosphate + 7,8-dihydropteroylglutamate
show the reaction diagram
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
carboxamide formation
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carboxylic acid amide formation
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-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Folate biosynthesis
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Metabolic pathways
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tetrahydrofolate biosynthesis
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tetrahydrofolate metabolism
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SYSTEMATIC NAME
IUBMB Comments
7,8-dihydropteroate:L-glutamate ligase (ADP-forming)
In some bacteria, a single protein catalyses both this activity and that of EC 6.3.2.17, tetrahydrofolate synthase [2], the combined activity of which leads to the formation of the coenzyme polyglutamated tetrahydropteroate (H4PteGlun), i.e. various tetrahydrofolates. In contrast, the activities are located on separate proteins in most eukaryotes studied to date [3]. This enzyme is reponsible for attaching the first glutamate residue to dihydropteroate to form dihydrofolate and is present only in those organisms that have the ability to synthesize tetrahydrofolate de novo, e.g. plants, most bacteria, fungi and protozoa [3].
CAS REGISTRY NUMBER
COMMENTARY hide
37318-62-0
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
ecotype Wassilewskija
Q8W041
SwissProt
Manually annotated by BRENDA team
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Manually annotated by BRENDA team
enzyme posseses both dihydrofolate synthetase activity and folylpolyglutamate synthetase activity
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
gene folC
A1KLE3
UniProt
Manually annotated by BRENDA team
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Manually annotated by BRENDA team
gene folC
UniProt
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
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SwissProt
Manually annotated by BRENDA team
CUY13 strain
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Manually annotated by BRENDA team
Serratia indica
IFO 3759
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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
malfunction
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ATP + 7,8-dihydropteroate + L-Glu
?
show the reaction diagram
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enzyme of 5,6,7,8-tetrahydropteroyl-Glun synthesis pathway
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-
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ATP + 7,8-dihydropteroate + L-Glu
ADP + phosphate + 7,8-dihydrofolate
show the reaction diagram
ATP + 7,8-dihydropteroate + L-glutamate
ADP + phosphate + 7,8-dihydropteroylglutamate
show the reaction diagram
ATP + L-glutamate + 7,8-dihydropteroate
ADP + phosphate + 7,8-dihydropteroylglutamate
show the reaction diagram
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assay at pH 10, 37C
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-
?
dATP + 7,8-dihydropteroate + L-Glu
dADP + phosphate + 7,8-dihydrofolate
show the reaction diagram
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40.5% of the activity relative to ATP
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GTP + 7,8-dihydropteroate + L-Glu
GDP + phosphate + 7,8-dihydrofolate
show the reaction diagram
ITP + 7,8-dihydropteroate + L-Glu
IDP + phosphate + 7,8-dihydrofolate
show the reaction diagram
UTP + 7,8-dihydropteroate + L-Glu
UDP + phosphate + 7,8-dihydrofolate
show the reaction diagram
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79.8% of the activity relative to ATP
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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
ATP + 7,8-dihydropteroate + L-Glu
?
show the reaction diagram
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enzyme of 5,6,7,8-tetrahydropteroyl-Glun synthesis pathway
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-
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ATP + 7,8-dihydropteroate + L-glutamate
ADP + phosphate + 7,8-dihydropteroylglutamate
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Fe2+
Serratia indica
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divalent cation requirement is satisfied by Mg2+, Mn2+ or Fe2+
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-[[(4-[[(2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl]amino]phenyl)(hydroxy)phosphoryl]methyl]pentanedioic acid
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; selective for dihydrofolate synthase activity
2-[[[(3S)-3-[(4-[[(2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl]amino]benzoyl)amino]-3-carboxypropyl](hydroxy)phosphoryl]methyl]pentanedioic acid
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7,8-dihydrofolate
Dihydro-10-carbonylfolic acid
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Dihydro-10-carbonylpteroic acid
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Dihydro-10-thiofolic acid
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Dihydro-10-thiopteroic acid
Dihydrofolate derivatives
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dihydrofolic acid
Dihydrohomopteroic acid
Dihydroisopteroic acid
Dihydromonopteroic acid
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Dihydropterin-sulfonamides
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Dihydropteroate derivatives
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p-hydroxymercuribenzoate
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Tetrahydrohomopteroate
Serratia indica
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additional information
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00025 - 0.035
7,8-dihydropteroate
0.0069 - 0.29
ATP
0.00088
dihydropteroate
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; pH 10.0, 37C
1.5 - 3.9
Glu
0.00597
glutamate
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0.006 - 9.1
L-Glu
0.0029 - 0.0081
MgATP2-
additional information
additional information
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non-standard kinetics at high glutamate and ATP concentrations, being partially inhibited by increasing concentrations of its principal substrate, dihydropteroate. Binding of dihydropteroate to the catalytic and inhibitory sites exhibits dissociation constants of 0.50 microM and 1.25 microM, respectively. Under lower co-substrate concentrations, data fit the Michaelis-Menten equation
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00014
2-[[(4-[[(2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl]amino]phenyl)(hydroxy)phosphoryl]methyl]pentanedioic acid
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; pH 10.0, 37C
0.00169
2-[[[(3S)-3-[(4-[[(2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl]amino]benzoyl)amino]-3-carboxypropyl](hydroxy)phosphoryl]methyl]pentanedioic acid
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IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.000041
2-[[(4-[[(2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl]amino]phenyl)(hydroxy)phosphoryl]methyl]pentanedioic acid
Plasmodium falciparum
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.007
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0.0155
Serratia indica
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additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
9
Serratia indica
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pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5 - 11
8 - 9.5
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8: about 25% of maximal activity, 9.5: about 85% of maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Escherichia coli (strain K12)
Escherichia coli (strain K12)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
47000
Serratia indica
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gel filtration
51000
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gel filtration
56000
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gel filtration
62000
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1 * 62400, calculated for His-tagged recombinant protein, 1 * 62000, SDS-PAGE
62400
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1 * 62400, calculated for His-tagged recombinant protein, 1 * 62000, SDS-PAGE
90000
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gel filtration, His-tagged recombinant protein
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
Q8W041
x * 53000, SDS-PAGE
monomer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
hanging drop method in 1.2-1.7 M ammonium sulfate
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
70
Serratia indica
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30 min, almost complete loss of activity
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
enzyme which is extracted from the isolated mitochondria is relatively stable in comparison with that extracted from whole cells
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relatively stable to trypsin pretreatment and relatively large amounts of protease and a temperature of 37C are required for digestion
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, unstable
Serratia indica
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-60C, stable
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4C, 50% loss of activity after 6 weeks
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stable in the form of an ammonium sulfate precipitate in an ice bath for 3 months
Serratia indica
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purification of a histidine-tagged recombinant enzyme using metal-affinity chromatography
recombinant wild-type and mutant enzymes with maltose binding protein-tag linked by a factor Xa cleavage site from Escherichia coli strain BL21(DE3) by amylose affinity chromatography, tag cleavage by faxtor Xa, dialysis, and anion exchange chromatography
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
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expression in Escherichia coli and Saccharomyces cerevisiae
expression in Escherichia coli BL21 cells; expression of His-tagged protein in Escherichia coli
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expression of a histidine-tagged recombinant enzyme in a Saccharomyces cerevisiae lacking a DHFS functional gene
gene folC, cloning and expression of wild-type and mutant enzymes with maltose binding protein-tag linked by a factor Xa cleavage site in Escherichia coli strain BL21(DE3)
Pneumocystis carinii DHFS expressed from a plasmid functionally complements a Saccharomyces cerevisiae mutant with no DHFS
the folC gene from Escherichia coli is cloned in plasmid pET29. The resulting plasmid, pVRC1432, is transferred into the Escherichia coli BL21 strain
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
expressed as single bifunctional protein with folypolyglutamate synthase, EC 6.3.2.17
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A183P
A1KLE3
naturally occuring conserved mutation, the mutant shows reduced enzyme activity
R49W
A1KLE3
naturally occuring conserved mutation, the mutant shows reduced enzyme activity
D112A
naturally occuring mutation
E153A
naturally occuring conserved mutation, the mutant shows reduced enzyme activity
E153G
naturally occuring conserved mutation
E40G
naturally occuring mutation
F152L
naturally occuring conserved mutation
F152S
naturally occuring conserved mutation
G111S
naturally occuring mutation
I43A
naturally occuring mutation
I43T
naturally occuring mutation, the mutant shows reduced enzyme activity
N73S
naturally occuring conserved mutation
R410W
naturally occuring mutation
S150G
naturally occuring conserved mutation
S150R
naturally occuring conserved mutation
E153A
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naturally occuring conserved mutation, the mutant shows reduced enzyme activity
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E153G
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naturally occuring conserved mutation
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R410W
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naturally occuring mutation
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S150R
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naturally occuring conserved mutation
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additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
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a simple radioassay for dihydrofolate synthetase activity and its application to an inhibition study of new pteroate analogs
biotechnology
medicine
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