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Information on EC 2.1.1.148 - thymidylate synthase (FAD) and Organism(s) Thermotoga maritima and UniProt Accession Q9WYT0
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2.1.1.148 thymidylate synthase (FAD)IUBMB Comments
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.
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Word Map
- 2.1.1.148
- medicine
- tuberculosis
- dtmp
- synthases
- thymidine
- dihydrofolate
- maritima
- bursaria
- 2'-deoxyuridine
-
paramecium
-
folate-dependent
- chlorella
-
5-substituted
-
virus-1
-
ch2h4folate
-
2'-deoxythymidine-5'-monophosphate
-
flavoenzyme
- drug development
The taxonomic range for the selected organisms is: Thermotoga maritima
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Synonyms
flavin-dependent thymidylate synthase, thymidylate synthase thyx, thymidylate synthase x, a674r, thymidylate synthase complementing protein, flavin dependent thymidylate synthase, flavin-dependent thymidylate synthase x, flavin-dependent ts, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
Thy1
-
-
-
-
thymidylate synthase complementing protein
-
-
-
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ThyX
TSCP
-
-
-
-
ThyX
-
-
-
-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
5,10-methylenetetrahydrofolate + dUMP + NADPH + H+ = dTMP + tetrahydrofolate + NADP+
a hydride equivalent is transferred from the reduced flavin cofactor directly to the uracil ring, followed by an isomerization of the intermediate to form the product, 2'-deoxythymidine-5'-monophosphate
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
methyl group transfer
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-
-
-
PATHWAY SOURCE
PATHWAYS
BRENDA
-
-
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
9031-61-2
-
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
5,10-methylenetetrahydrofolate + dUMP + NADPH + H+
dTMP + tetrahydrofolate + NADP+
-
-
-
?
5,10-methylenetetrahydrofolate + dUMP + FADH2
dTMP + tetrahydrofolate + FAD
-
dUMP dependent lag-phase for the single turnover reduction of FDTS bound FAD by NADPH
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-
?
5,10-methylenetetrahydrofolate + dUMP + FADH2 + O2
dTMP + tetrahydrofolate + FAD + H2O2
-
-
-
-
?
5,10-methylenetetrahydrofolate + dUMP + FADH2
dTMP + tetrahydrofolate + FAD
-
-
?
5,10-methylenetetrahydrofolate + dUMP + FADH2
dTMP + tetrahydrofolate + FAD
-
-
?
5,10-methylenetetrahydrofolate + dUMP + FADH2
dTMP + tetrahydrofolate + FAD
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-
-
?
additional information
?
-
the enzyme is essential to DNA replication
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-
?
additional information
?
-
-
the enzyme is essential to DNA replication
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-
?
additional information
?
-
flavin-dependent thymidylate synthase can function as an oxidase, which catalyzes the reduction of O2 to H2O2, using reduced NADPH or other reducing agents and a sequential kinetic mechanism of substrate binding. Tetrahydrofolate competitively inhibits the oxidase activity, which indicates that tetrahydrofolate and O2 compete for the same reduced and dUMP-activated enzymatic complex
-
-
?
additional information
?
-
an initial activation of the pyrimidine substrate by reduced flavin is required for catalysis. A H/D exchange at C5 of dUMP in D2O requires the flavin to be reduced, as observed experimentally. Chemical trapping of reaction intermediates shows the unusual intermediates of further steps
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-
?
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
5,10-methylenetetrahydrofolate + dUMP + FADH2
dTMP + tetrahydrofolate + FAD
-
-
-
?
5,10-methylenetetrahydrofolate + dUMP + FADH2 + O2
dTMP + tetrahydrofolate + FAD + H2O2
-
-
-
-
?
additional information
?
-
the enzyme is essential to DNA replication
-
-
?
additional information
?
-
-
the enzyme is essential to DNA replication
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
NADPH
the pro-R hydride of NADPH and not the R6 hydride of the tetrahydropterin is the reducing agent. The stereospecificity for the pro-R NADPH oxidation is not absolute. The hydride is transferred to FAD, which is the rate-limiting step of the catalytic cascade
FAD
even though FAD is very well bound to three subunits, FAD interactions are not an absolute requirement for tetramer stabilization
FAD
the pro-R hydride of NADPH and not the R6 hydride of the tetrahydropterin is the reducing agent. The stereospecificity for the pro-R NADPH oxidation is not absolute. The hydride is transferred to FAD, which is the rate-limiting step of the catalytic cascade
FAD
presence of FAD additionally stabilizes the protein against thermal denaturation by 4.9 degrees. Binding of dUMP to FAD-loaded proteins stabilizes further. ThyX binds FAD with a low micromolar binding affinity in a process characterized by a favorable entropy change
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
(2R)-2-[(3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-yl)sulfanyl]propanoic acid
6% inhibition at 0.1 mM
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
NADPH
-
inhibits FDTS at high concentrations at all temperatures, NADPH may not be the natural reducing agent of FDTS
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.0046
dUMP
pH not specified in the publication, temperature not specified in the publication
0.13
NADPH
pH not specified in the publication, temperature not specified in the publication
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
SwissProt
PDB
SCOP
CATH
UNIPROT
ORGANISM
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
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
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
structure of ThyX in complex with nonsubstrate analog inhibitor (2R)-2-[(3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-yl)sulfanyl]propanoic acid
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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
Y91F
similar to wild-type, at saturating FAD conditions dUMP binding to the protein/FAD complex leads to additional stabilization by about 7 degrees
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
medicine
the unique mechanism of FDTS makes it an attractive target for antibiotic drug development
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Montfort, W.R.
Complementing thymidylate synthase
Structure
11
607-608
2003
Dictyostelium discoideum, Thermotoga maritima (Q9WYT0)
Mathews, I.I.; Deacon, A.M.; Canaves, J.M.; McMullan, D.; Lesley, S.A.; Agarwalla, S.; Kuhn, P.
Functional analysis of substrate and cofactor complex structures of a thymidylate synthase-complementing protein
Structure
11
677-690
2003
Bacillus anthracis, Borreliella burgdorferi, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia trachomatis, Clostridioides difficile, Clostridium botulinum, Clostridium perfringens, Corynebacterium diphtheriae, Dictyostelium discoideum, Ehrlichia chaffeensis, Helicobacter pylori, Helicobacter pylori (Q9ZJ93), Mycobacterium avium, Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium tuberculosis variant bovis, Rhodococcus sp., Rickettsia conorii, Rickettsia prowazekii, Thermotoga maritima (Q9WYT0), Thermotoga maritima DSM 3109 (Q9WYT0), Treponema denticola, Treponema pallidum
Leduc, D.; Graziani, S.; Meslet-Cladiere, L.; Sodolescu, A.; Liebl, U.; Myllykallio, H.
Two distinct pathways for thymidylate (dTMP) synthesis in (hyper)thermophilic bacteria and archaea
Biochem. Soc. Trans.
32
231-235
2004
Aeropyrum pernix, Aquifex aeolicus, no activity in Archaeoglobus fulgidus, no activity in Methanocaldococcus jannaschii, no activity in Methanopyrus kandleri, no activity in Methanopyrus kandleri AV19, no activity in Methanothermobacter thermoautotrophicus, Pyrobaculum aerophilum, Pyrococcus abyssi, Pyrococcus furiosus, Pyrococcus horikoshii, Saccharolobus solfataricus, Sulfurisphaera tokodaii, Thermoplasma acidophilum, Thermoplasma volcanium, Thermotoga maritima
Agrawal, N.; Lesley, S.A.; Kuhn, P.; Kohen, A.
Mechanistic studies of a flavin-dependent thymidylate synthase
Biochemistry
43
10295-10301
2004
Thermotoga maritima (Q9WYT0), Thermotoga maritima, Thermotoga maritima DSM 3109 (Q9WYT0)
Chernyshev, A.; Fleischmann, T.; Kohen, A.
Thymidyl biosynthesis enzymes as antibiotic targets
Appl. Microbiol. Biotechnol.
74
282-289
2007
Bacillus anthracis, Chlamydia trachomatis, Clostridium botulinum, Helicobacter pylori, Mycobacterium leprae, Mycobacterium tuberculosis (P9WG57), Mycobacterium tuberculosis H37Rv (P9WG57), no activity in Homo sapiens, Paramecium bursaria Chlorella virus-1, Pyrococcus furiosus, Rickettsia sp., Thermotoga maritima, Treponema palladium
Mason, A.; Agrawal, N.; Washington, M.T.; Lesley, S.A.; Kohen, A.
A lag-phase in the reduction of flavin dependent thymidylate synthase (FDTS) revealed a mechanistic missing link
Chem. Commun. (Camb. )
16
1781-1783
2006
Thermotoga maritima
Chernyshev, A.; Fleischmann, T.; Koehn, E.M.; Lesley, S.A.; Kohen, A.
The relationships between oxidase and synthase activities of flavin dependent thymidylate synthase (FDTS)
Chem. Commun. (Camb. )
27
2861-2863
2007
Thermotoga maritima
Wang, Z.; Chernyshev, A.; Koehn, E.M.; Manuel, T.D.; Lesley, S.A.; Kohen, A.
Oxidase activity of a flavin-dependent thymidylate synthase
FEBS J.
276
2801-2810
2009
Thermotoga maritima (Q9WYT0)
Koehn, E.M.; Fleischmann, T.; Conrad, J.A.; Palfey, B.A.; Lesley, S.A.; Mathews, I.I.; Kohen, A.
An unusual mechanism of thymidylate biosynthesis in organisms containing the thyX gene
Nature
458
919-923
2009
Thermotoga maritima (Q9WYT0)
Mishanina, T.V.; Koehn, E.M.; Conrad, J.A.; Palfey, B.A.; Lesley, S.A.; Kohen, A.
Trapping of an intermediate in the reaction catalyzed by flavin-dependent thymidylate synthase
J. Am. Chem. Soc.
134
4442-4448
2012
Thermotoga maritima (Q9WYT0), Thermotoga maritima ATCC 43589 (Q9WYT0)
Mishanina, T.V.; Corcoran, J.M.; Kohen, A.
Substrate activation in flavin-dependent thymidylate synthase
J. Am. Chem. Soc.
136
10597-10600
2014
Thermotoga maritima (Q9WYT0), Thermotoga maritima ATCC 43589 (Q9WYT0)
Mathews, I.I.
Flavin-dependent thymidylate synthase as a drug target for deadly microbes: mutational study and a strategy for inhibitor design
J. Bioterror Biodef.
Suppl 12
004
2013
Thermotoga maritima (Q9WYT0), Thermotoga maritima ATCC 43589 (Q9WYT0)
Krumova, S.; Todinova, S.; Tileva, M.; Bouzhir-Sima, L.; Vos, M.H.; Liebl, U.; Taneva, S.G.
Thermal stability and binding energetics of thymidylate synthase ThyX
Int. J. Biol. Macromol.
91
560-567
2016
Paramecium bursaria Chlorella virus 1 (O41156), Paramecium bursaria Chlorella virus 1, Thermotoga maritima (Q9WYT0), Thermotoga maritima, Thermotoga maritima DSM 3109 (Q9WYT0)
Luciani, R.; Saxena, P.; Surade, S.; Santucci, M.; Venturelli, A.; Borsari, C.; Marverti, G.; Ponterini, G.; Ferrari, S.; Blundell, T.L.; Costi, M.P.
Virtual screening and X-ray crystallography identify non-substrate analog inhibitors of flavin-dependent thymidylate synthase
J. Med. Chem.
59
9269-9275
2016
Thermotoga maritima (Q9WYT0), Thermotoga maritima DSM 3109 (Q9WYT0)
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