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
tetrahydrofolate biosynthesis
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tetrahydrofolate metabolism
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Folate biosynthesis
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Metabolic pathways
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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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-
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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
Escherichia coli B / ATCC 11303
B
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Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
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-
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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
Mycobacterium tuberculosis ATCC 25618 / H37Rv
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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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-
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
metabolism
physiological function
additional information
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, 37°C
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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, 37°C
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, 37°C
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
UNIPROT
ORGANISM
Escherichia coli (strain K12);
Q8D0U0
Yersinia pestis;
A0A0H2YK03
Yersinia pestis bv. Antiqua (strain Nepal516);
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 37°C are required for digestion
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20°C, unstable
Serratia indica
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-60°C, stable
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4°C, 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 dhfs, DNA and amino acid sequence determination and analysis, DNA is isolated from a bronchoalveolar lavage fluid sample of a patient, sequence comparisons, subcloning in Escherichia coli strain DH5alpha, heterologous expression of the enzyme can rescue of the null allele of the orthologous gene of Saccharomyces cerevisiae strain LCY1, functional complementation of the strain lacking the DHFS encoding gene FOL3
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)
gene folC, DNA and amino acid sequence determination and analysis, genetic structure and sequence comparisons, genotyping of 254 clinical isolates, phylogenetic analysis
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
A132E
naturally occuring mutation in enzyme FolC in clinical isolates of drug-susceptible Mycobacterium tuberculosis
A171A
naturally occuring mutation in enzyme FolC in clinical isolates of drug-susceptible Mycobacterium tuberculosis
A420V
naturally occuring mutation in enzyme FolC in clinical isolates of both drug-resistant and drug-susceptible Mycobacterium tuberculosis
A457V
naturally occuring mutation in enzyme FolC in clinical isolates of drug-resistant Mycobacterium tuberculosis
D111A
naturally occuring mutation in enzyme FolC in clinical isolates of drug-resistant Mycobacterium tuberculosis
D112A
naturally occuring mutation
D135A
naturally occuring mutation in enzyme FolC in clinical isolates of drug-resistant Mycobacterium tuberculosis
D324G
naturally occuring mutation in enzyme FolC in clinical isolates of drug-susceptible Mycobacterium tuberculosis
D384A
naturally occuring mutation in enzyme FolC in clinical isolates of drug-susceptible Mycobacterium tuberculosis
E40K
naturally occuring mutation in enzyme FolC in clinical isolates of drug-resistant Mycobacterium tuberculosis
E434Q
naturally occuring mutation in enzyme FolC in clinical isolates of drug-resistant Mycobacterium tuberculosis
F152L
naturally occuring conserved mutation
F152S
naturally occuring conserved mutation
F461F
naturally occuring mutation in enzyme FolC in clinical isolates of drug-susceptible Mycobacterium tuberculosis
G111S
naturally occuring mutation
G112S
naturally occuring mutation in enzyme FolC in clinical isolates of drug-resistant Mycobacterium tuberculosis
G284G
naturally occuring mutation in enzyme FolC in clinical isolates of drug-resistant Mycobacterium tuberculosis
G422G
naturally occuring mutation in enzyme FolC in clinical isolates of drug-resistant Mycobacterium tuberculosis
I43F
naturally occuring mutation in enzyme FolC in clinical isolates of drug-resistant Mycobacterium tuberculosis
I43S
naturally occuring mutation in enzyme FolC in clinical isolates of drug-resistant Mycobacterium tuberculosis
I43V
naturally occuring mutation in enzyme FolC in clinical isolates of drug-resistant Mycobacterium tuberculosis
N73S
naturally occuring conserved mutation
P21L
naturally occuring mutation in enzyme FolC in clinical isolates of drug-susceptible Mycobacterium tuberculosis
P356L
naturally occuring mutation in enzyme FolC in clinical isolates of drug-susceptible Mycobacterium tuberculosis
P8P
naturally occuring mutation in enzyme FolC in clinical isolates of drug-susceptible Mycobacterium tuberculosis
P9P
naturally occuring mutation in enzyme FolC in clinical isolates of drug-susceptible Mycobacterium tuberculosis
R268R
naturally occuring mutation in enzyme FolC in clinical isolates of drug-resistant Mycobacterium tuberculosis
R49G
naturally occuring mutation in enzyme FolC in clinical isolates of drug-susceptible Mycobacterium tuberculosis
S150R
naturally occuring conserved mutation
S335I
naturally occuring mutation in enzyme FolC in clinical isolates of drug-resistant Mycobacterium tuberculosis
V256A
naturally occuring mutation in enzyme FolC in clinical isolates of drug-resistant Mycobacterium tuberculosis
D111A
Mycobacterium tuberculosis ATCC 25618 / H37Rv
-
naturally occuring mutation in enzyme FolC in clinical isolates of drug-resistant Mycobacterium tuberculosis
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D384A
Mycobacterium tuberculosis ATCC 25618 / H37Rv
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naturally occuring mutation in enzyme FolC in clinical isolates of drug-susceptible Mycobacterium tuberculosis
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E153A
Mycobacterium tuberculosis ATCC 25618 / H37Rv
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naturally occuring mutation in enzyme FolC in clinical isolates of drug-resistant Mycobacterium tuberculosis
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E153G
Mycobacterium tuberculosis ATCC 25618 / H37Rv
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naturally occuring mutation in enzyme FolC in clinical isolates of drug-resistant Mycobacterium tuberculosis
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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
-
a simple radioassay for dihydrofolate synthetase activity and its application to an inhibition study of new pteroate analogs
biotechnology
drug development
medicine
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