Information on EC 2.1.1.148 - thymidylate synthase (FAD)

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
2.1.1.148
-
RECOMMENDED NAME
GeneOntology No.
thymidylate synthase (FAD)
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
5,10-methylenetetrahydrofolate + dUMP + NADPH + H+ = dTMP + tetrahydrofolate + NADP+
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
methyl group transfer
-
-
-
-
reductive methylation
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Metabolic pathways
-
-
One carbon pool by folate
-
-
pyrimidine deoxyribonucleotides de novo biosynthesis III
-
-
pyrimidine metabolism
-
-
Pyrimidine metabolism
-
-
SYSTEMATIC NAME
IUBMB Comments
5,10-methylenetetrahydrofolate,FADH2:dUMP C-methyltransferase
Contains FAD. All thymidylate synthases catalyse a reductive methylation involving the transfer of the methylene group of 5,10-methylenetetrahydrofolate to the C5 position of dUMP and a two electron reduction of the methylene group to a methyl group. Unlike the classical thymidylate synthase, ThyA (EC 2.1.1.45), which uses folate as both a 1-carbon donor and a source of reducing equivalents, this enzyme uses a flavin coenzyme as a source of reducing equivalents, which are derived from NADPH.
CAS REGISTRY NUMBER
COMMENTARY hide
9031-61-2
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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-
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Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
Borrelia burgdorferi
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
enzyme found in three different species
-
-
Manually annotated by BRENDA team
no activity in Archaeoglobus fulgidus
-
-
-
Manually annotated by BRENDA team
no activity in Corynebacterium glutamicum strain NCHU 87078
-
-
-
Manually annotated by BRENDA team
no activity in Homo sapiens
-
-
-
Manually annotated by BRENDA team
no activity in Methanocaldococcus jannaschii
-
-
-
Manually annotated by BRENDA team
no activity in Methanopyrus kandleri
AV19
-
-
Manually annotated by BRENDA team
no activity in Methanopyrus kandleri AV19
AV19
-
-
Manually annotated by BRENDA team
no activity in Methanothermobacter thermoautotrophicus
-
-
-
Manually annotated by BRENDA team
Paramecium bursaria Chlorella virus-1
-
-
-
Manually annotated by BRENDA team
strain MT1131
-
-
Manually annotated by BRENDA team
strain MT1131
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
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SwissProt
Manually annotated by BRENDA team
Thermotoga maritima ATCC 435890
-
SwissProt
Manually annotated by BRENDA team
TM0449
SwissProt
Manually annotated by BRENDA team
Treponema palladium
-
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
5,10-methylenetetrahydrofolate + BrdUMP + FADH2
?
show the reaction diagram
-
-
-
-
?
5,10-methylenetetrahydrofolate + dUMP + FADH2
dTMP + tetrahydrofolate + FAD
show the reaction diagram
5,10-methylenetetrahydrofolate + dUMP + FADH2 + O2
dTMP + tetrahydrofolate + FAD + H2O2
show the reaction diagram
-
-
-
-
?
5,10-methylenetetrahydrofolate + dUMP + FMNH2
dTMP + tetrahydrofolate + FMN
show the reaction diagram
5,10-methylenetetrahydrofolate + dUMP + NADPH
dTMP + tetrahydrofolate + NADP
show the reaction diagram
additional information
?
-
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
5,10-methylenetetrahydrofolate + dUMP + FADH2
dTMP + tetrahydrofolate + FAD
show the reaction diagram
5,10-methylenetetrahydrofolate + dUMP + FADH2 + O2
dTMP + tetrahydrofolate + FAD + H2O2
show the reaction diagram
-
-
-
-
?
5,10-methylenetetrahydrofolate + dUMP + NADPH
dTMP + tetrahydrofolate + NADP
show the reaction diagram
-
Rhodobacter capsulatus ThyX is required for de novo thymidylate synthesis
-
-
?
additional information
?
-
Q9WYT0
the enzyme is essential to DNA replication
-
-
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NAD(P)H
NADP+
-
expulsion of the cofactor FAD by NADP+, no anticipated ThyX-FAD-BrdUMP-NADP+ quaternary complex, but a ThyX-NADP+ binary complex
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Mn2+
0.5 mM enhances the activity
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(heptanoyl[[(4R)-2-phenyl-3-(phenylcarbonyl)-1,3-thiazolidin-4-yl]methyl] amino)acetic acid
-
10-methyl-5,8-dideazafolate
Paramecium bursaria Chlorella virus-1
-
0.2 mM, 90% inhibition
10-propargylfolate
Paramecium bursaria Chlorella virus-1
-
0.2 mM, 85% inhibition
5,10-methylenetetrahydrofolate
Paramecium bursaria Chlorella virus-1
-
-
5-fluoro-2'-deoxyuridine 5'-monophosphate
-
FdUMP
5-fluorodeoxyuridine
Paramecium bursaria Chlorella virus-1
-
0.05 mM, 95% inhibition
5-undecyloxymethyl-2'-deoxyuridine 5'-monophosphate
-
no activity against classical thymidylate synthase ThyA, EC 2.1.1.45
-
8-aminopurinone deoxyribonucleoside 5'-phosphate
Paramecium bursaria Chlorella virus-1
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0.05 mM, 50% inhibition
diethyldicarbonate
Paramecium bursaria Chlorella virus-1
-
decreases ThyX deprotonation activity at least 20fold, is partially reversible with hydroxylamine treatment
ethyl (4R)-2-phenyl-3-([(1-(3-2,2,2-trifluoroacetamido)propyl)-1H-1,2,3-triazol-4-yl]carbonyl)-1,3-thiazolidine-4-carboxylate
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ethyl (4R)-2-phenyl-3-[[1-(2-thiophen-3-ylethyl)-1H-1,2,3-triazol-4-yl]carbonyl]-1,3-thiazolidine-4-carboxylate
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ethyl (4R)-3-([1-[2-(4-fluorophenyl)ethyl]-1H-1,2,3-triazol-4-yl]carbonyl)-2-phenyl-1,3-thiazolidine-4-carboxylate
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ethyl (4R)-3-[[1-(7-hydroxyheptyl)-1H-1,2,3-triazol-4-yl]carbonyl]-2-phenyl-1,3-thiazolidine-4-carboxylate
-
tetrahydrofolate
competitively inhibits the oxidase activity of the enzyme, which indicates that tetrahydrofolate and O2 compete for the same reduced and dUMP-activated enzymatic complex
additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
FAD
-
increases activity
FMN
-
increases activity
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.004 - 0.35
5,10-methylenetetrahydrofolate
0.00083 - 0.0153
dUMP
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0038 - 0.14
dUMP
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00013
(heptanoyl[[(4R)-2-phenyl-3-(phenylcarbonyl)-1,3-thiazolidin-4-yl]methyl] amino)acetic acid
-
0.0001
5-fluoro-2'-deoxyuridine 5'-monophosphate
-
inhibited both ThyA and ThyX
0.000057
ethyl (4R)-2-phenyl-3-([(1-(3-2,2,2-trifluoroacetamido)propyl)-1H-1,2,3-triazol-4-yl]carbonyl)-1,3-thiazolidine-4-carboxylate
-
0.001
ethyl (4R)-2-phenyl-3-[[1-(2-thiophen-3-ylethyl)-1H-1,2,3-triazol-4-yl]carbonyl]-1,3-thiazolidine-4-carboxylate
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0.002
ethyl (4R)-3-([1-[2-(4-fluorophenyl)ethyl]-1H-1,2,3-triazol-4-yl]carbonyl)-2-phenyl-1,3-thiazolidine-4-carboxylate
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0.0035
ethyl (4R)-3-[[1-(7-hydroxyheptyl)-1H-1,2,3-triazol-4-yl]carbonyl]-2-phenyl-1,3-thiazolidine-4-carboxylate
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IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.016 - 0.048
5,10-methylenetetrahydrofolate
0.0083
5-undecyloxymethyl-2'-deoxyuridine 5'-monophosphate
Mycobacterium tuberculosis
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pH not specified in the publication, temperature not specified in the publication
-
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
75
optimum RNA-binding activity
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
PDB
SCOP
CATH
ORGANISM
UNIPROT
Corynebacterium glutamicum (strain ATCC 13032 / DSM 20300 / JCM 1318 / LMG 3730 / NCIMB 10025)
Corynebacterium glutamicum (strain ATCC 13032 / DSM 20300 / JCM 1318 / LMG 3730 / NCIMB 10025)
Helicobacter pylori (strain ATCC 700392 / 26695)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
23800
-
SDS-PAGE
31000
-
SDS-PAGE
31500
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calculated from DNA sequence
104000
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gel filtration
111000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homotetramer
tetramer
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
proteolytic modification
-
the ThyX sequence has an intein in itsThyX motif that does protein spicing and a group II intron, suggesting a hot spot for these self-splicing mobile elements
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
hanging drop vapor diffusion method, using 0.2 M sodium chloride, 0.1 M MES, pH 6.0, and 20% (w/v) PEG2000 monomethyl ether
in complex with FAD and dUMP
-
by the sitting-drop vapor diffusion method, in the presence of the cofactor FAD and the substrate analog BrdUMP, at 2.0 A resolution
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I65M/L175M double mutant in the presence of FAD and BrdUMP, by the sitting-drop vapor-diffusion method, to 2.0 A resolution
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molecular modeling of the complex with inhibitor 5-undecyloxymethyl-2'-deoxyuridine 5'-monophosphate. Inhibitor contacts mainly with amino acid residues of subunits A and C. The hydrophobic substituent at the C-5 position of nucleic base occupies active site mainly due to lipophilic interactions. Several amino acids of the active site such as Arg95, Gln103, Arg107, Arg172, and Gln106 being involved in the interaction with natural substrate dUMP contribute for affinity due to hydrogen bonds
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crystal structure of the ThyX protein complexed to its FAD cofactor and solved by molecular replacement, to 2.3 A resolution, crystals belong to the P21212 space group with predicted two molecules per asymmetric unit and a solvent content of 51%
Paramecium bursaria Chlorella virus-1
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crystal structure shows certain small structural differences in the active site when compared with either bacterial FDTS
Paramecium bursaria Chlorella virus-1
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crystal structure shows no structural similarity with “classical” TS
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mutant H53D, in complex with the methyl donor, CH2H4 folate. The substrate-binding loop can be stabilized in two conformations and this affects the binding of the molecules at the substrate binding site. The isoalloxazine (flavin) ring of FAD binds in a big pocket that tolerates large movements of the isoalloxazine ring. The isoalloxazine ring is able to rotate in the binding pocket and utilize same face of the ring to bind to substrate and cofactors
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mutants S88A and S88C, to 1.95 and 2.05 A resolution, respectively. Structure reveals minimlas changes in folding and active site conformation compared to wild-type. There is no covalent bond between the cysteine and dUMP in the crystal
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-80°C, 10 mM Tris-HCl buffer, pH 7.2, 50 mM NaCl, 2 mM DTT, 100 mM FAD
-
-80°C, 50 mM HEPES buffer, pH 7.0, 10% glycerol
Paramecium bursaria Chlorella virus-1
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
by nickel chromatography and gel filtration, more than 95% pure
Paramecium bursaria Chlorella virus-1
-
from transformed E. coli
-
from transformed E. coli using His-tag
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nickel affinity and ion-exchange chromatography
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nickel chelating column chromatography, GSH-Sepharose column chromatography, and Superdex 200 gel filtration
on Ni-NTA column
-
to near homogeneity, by heat treatment at 85°C for 15 min, followed by Ni2+ column chromatography
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
complemetation of Escherichia coli thyA deletion mutant, transformants are able to grow in the absence of thymidine under oxygen limited conditions
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complemetation of Escherichia coli, transformants are able to grow in the absence of thymidine
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complemetation of Escherichia coli, transformants are unable to grow in the absence of thymidine
complemetation of Haloferax volcanii thyA deletion mutant, transformants are able to grow in the absence of thymidine
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expressed in Escherichia coli M15 cells
expression in Escherichia coli
Paramecium bursaria Chlorella virus-1
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histidine-tagged Mycobacterium tuberculosis ThyX is overexpressed from BL21-DE3 pLysS Escherichia coli
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I65M/L175M double mutant expressed in Escherichia coli BL21 (DE3) pLysS strain as C-terminal His6-tagged protein
-
into the pET24d vector, overexpressed in Escherichia coli chi2913 strain, plasmid containing the I65M/L175M double mutant expressed in Escherichia coli BL21 (DE3) pLysS strain
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overexpression of the recombinant C-terminal His6-tagged protein in Escherichia coli strain BL21(DE3)
wild-type and mutant ThyX proteins expressed in either Escherichia coli DH5alpha (deltathyA) or BL21
Paramecium bursaria Chlorella virus-1
-
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
replacement of Escherichia coli thyA gene, EC 2.1.1.45, by thyX and expression under the native thyA promoter. In thymidine-deprived solid and liquid growth media, the mutant strain grows poorly compared with the wild-type strain and is impaired in DNA replication
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
H48Q
-
inactive mutant enzyme
H48Q/S84A
-
inactive mutant enzyme
H48Q/S84C
-
inactive mutant enzyme
R74A
-
the KM-value for dUMP is 5.1fold higher than the wild-type value, the turnover-number is 4.6fold lower than the wild-type value
R74K
-
the KM-value for dUMP is nearly identical to wild-type value, the turnover-number is 6.6fold lower than the wild-type value
S107A
-
no activity
S84A
-
the KM-value for dUMP is 5.3fold higher than the wild-type value, the turnover-number is 2.1fold lower than the wild-type value. Mutation abolishes thymidylate synthase activity in vivo
S84A/S85A
-
inactive mutant enzyme
S84C
-
mutation abolishes thymidylate synthase activity in vivo
S84Y
-
the KM-value for dUMP is 1.9fold higher than the wild-type value, the turnover-number is 1.6fold lower than the wild-type value
Y87F
-
the KM-value for dUMP is 1.8fold lower than the wild-type value, the turnover-number is nearly identical to the wild-type value
H69E
-
fails to complement the Escherichia coli chi2913 cells
I65M
-
no impaired enzyme activity, encodes proteins supporting the growth of Escherichia coli chi2913 strain
I65M/L175M
-
produces active ThyX enzyme
K165A
-
fails to complement the Escherichia coli chi2913 cells
L175M
-
no impaired enzyme activity, encodes proteins supporting the growth of Escherichia coli chi2913 strain
R168A
-
fails to complement the Escherichia coli chi2913 cells
R95A
-
fails to complement the Escherichia coli chi2913 cells
R95D
-
fails to complement the Escherichia coli chi2913 cells
R95K
-
supports growth of Escherichia coli chi2913 cells
S105E
-
fails to complement the Escherichia coli chi2913 cells
Y108F
-
complemens the growth of Escherichia coli chi2913 cells
H177K
Paramecium bursaria Chlorella virus-1
-
does not confer thymidine-independent growth to an Escherichia coli strain lacking thymidylate synthase ThyA, 9.5% oxidation activity
H177Q
Paramecium bursaria Chlorella virus-1
-
confers thymidine-independent growth to an Escherichia coli strain lacking thymidylate synthase ThyA, 31% oxidation activity
H53K
Paramecium bursaria Chlorella virus-1
-
does not confer thymidine-independent growth to an Escherichia coli strain lacking thymidylate synthase ThyA, produces insoluble protein
H53Q
Paramecium bursaria Chlorella virus-1
-
does not confer thymidine-independent growth to an Escherichia coli strain lacking thymidylate synthase ThyA, produces insoluble protein
H79K
Paramecium bursaria Chlorella virus-1
-
does not confer thymidine-independent growth to an Escherichia coli strain lacking thymidylate synthase ThyA
H79Q
Paramecium bursaria Chlorella virus-1
-
confers thymidine-independent growth to an Escherichia coli strain lacking thymidylate synthase ThyA, 94% oxidation activity
R182A
Paramecium bursaria Chlorella virus-1
-
does not confer thymidine-independent growth to an Escherichia coli strain lacking thymidylate synthase ThyA, does not copurify with oxidized FAD, but is able to oxidize NADPH in the presence of 0.4 mM FAD
R90A
Paramecium bursaria Chlorella virus-1
-
does not confer thymidine-independent growth to an Escherichia coli strain lacking thymidylate synthase ThyA, looses 44% of its oxidation activity and shows no measurable deprotonation activity
H53D
-
residue H53 is involved in folate binding. Crystal structures of the H53D-FAD and H53D-FAD-dUMP complexes
S88A
-
mutant retains activity. Residue S88 is not required for catalysis
S88C
-
mutant retains activity. Residue S88 is not required for catalysis
H53D
-
residue H53 is involved in folate binding. Crystal structures of the H53D-FAD and H53D-FAD-dUMP complexes
-
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
drug development
-
unexpected observation that NADP+ competes with both FAD and substrate for the binding site in ThyX and displaces both molecules from the active site, opens avenues for the design of tight-binding inhibitors of ThyX enzymes from a variety of organisms
medicine
additional information
Show AA Sequence (2304 entries)
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