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Information on EC 2.1.1.148 - thymidylate synthase (FAD) and Organism(s) Helicobacter pylori and UniProt Accession O26061

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EC Tree
     2 Transferases
         2.1 Transferring one-carbon groups
             2.1.1 Methyltransferases
                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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Helicobacter pylori
UNIPROT: O26061
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The taxonomic range for the selected organisms is: Helicobacter pylori
The enzyme appears in selected viruses and cellular organisms
Synonyms
flavin-dependent thymidylate synthase, thymidylate synthase thyx, a674r, thymidylate synthase x, thymidylate synthase complementing protein, flavin dependent thymidylate synthase, flavin-dependent thymidylate synthase x, flavin-dependent ts, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
complementing thymidylate synthase
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-
flavin-dependent thymidylate synthase
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-
Thy1
-
-
-
-
thymidylate synthase complementing protein
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-
-
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thymidylate synthase X
TSCP
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
methyl group transfer
-
-
-
-
PATHWAY SOURCE
PATHWAYS
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-
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
-
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 + dUMP + FADH2
dTMP + tetrahydrofolate + FAD
show the reaction diagram
5,10-methylenetetrahydrofolate + dUMP + FMNH2
dTMP + tetrahydrofolate + FMN
show the reaction diagram
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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
5,10-methylenetetrahydrofolate + dUMP + FADH2
dTMP + tetrahydrofolate + FAD
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
FADH2
FMNH2
activity is dependent on reduced flavin nucleotides
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-(furan-2-yl)-3-hydroxynaphthalene-1,4-dione
inhibits the NADPH oxidation and 2'-deoxythymidine-5'-monophosphate-forming activities of ThyX in vitro
2-hydroxy-3-(4-methoxyphenyl)naphthalene-1,4-dione
inhibits the NADPH oxidation and 2'-deoxythymidine-5'-monophosphate-forming activities of ThyX in vitro
2-hydroxy-3-propylnaphthalene-1,4-dione
inhibits the NADPH oxidation and 2'-deoxythymidine-5'-monophosphate-forming activities of ThyX in vitro, significant decrease of 1.22 log (17fold) in the colonization loads with Helicobacter pylori of mice treated with the molecule
2-[4-fluoro-3-(trifluoromethyl)phenyl]-3-hydroxynaphthalene-1,4-dione
inhibits the NADPH oxidation and 2'-deoxythymidine-5'-monophosphate-forming activities of ThyX in vitro
NADPH
excess NADPH decreases enzyme activity
additional information
-
functional evidence for active site location of tetrameric thymidylate synthase X at the interphase of three monomers. The active-site configurations of ThyX proteins, present in many human pathogenic bacteria, and of human thymidylate synthase A (EC 2.1.1.45) are different.
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
FAD
increases activity
FMN
increases activity
NADPH
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.35
5,10-methylenetetrahydrofolate
in 50 mM Tris-HCl pH 8.0, 10 mM MgCl2, 2 mM NADPH, 0.5 mM FAD, at 22°C
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
0.000028
2-(furan-2-yl)-3-hydroxynaphthalene-1,4-dione
pH 8.0, 37°C
0.000367
2-hydroxy-3-(4-methoxyphenyl)naphthalene-1,4-dione
pH 8.0, 37°C
0.000258
2-hydroxy-3-propylnaphthalene-1,4-dione
pH 8.0, 37°C
0.001
2-[4-fluoro-3-(trifluoromethyl)phenyl]-3-hydroxynaphthalene-1,4-dione
pH 8.0, 37°C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
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DNA replication speed in bacteria and archaea that contain the low-activity ThyX enzyme is up to 10fold decreased compared with species that contain the catalytically more efficient ThyA, EC 2.1.1.45. Both ThyX and ThyA participate in frequent reciprocal gene replacement events. The bacterial metabolism continues to modulate the size and composition of prokaryotic genomes. The increased kinetic efficiency of thymidylate synthesis may have contributed to extending the prokaryotic evolutionary potential
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
111000
gel filtration
25000
4 * 25000, SDS-PAGE
25020
4 * 25020, calculated from amino acid sequence
25066
4 * 25066, MALDI TOF mass spectrometry
31500
calculated from DNA sequence
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
-
x * 27168.2, wild-type enzyme, MALDI-TOF
homotetramer
tetramer
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
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
H48Q
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inactive mutant enzyme
H48Q/S84A
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inactive mutant enzyme
H48Q/S84C
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inactive mutant enzyme
R74A
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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
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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
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inactive mutant enzyme
S84C
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mutation abolishes thymidylate synthase activity in vivo
S84Y
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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
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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
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
from transformed E. coli using His-tag
nickel chelating column chromatography, GSH-Sepharose column chromatography, and Superdex 200 gel filtration
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
complemetation of Escherichia coli, transformants are able to grow in the absence of thymidine
expressed in Escherichia coli M15 cells
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Myllykallio, H.; Lipowski, G.; Leduc, D.; Filee, J.; Forterre, P.; Liebl, U.
An alternative flavin-dependent mechanism for thymidylate synthesis
Science
297
105-107
2002
Borreliella burgdorferi, Campylobacter jejuni, Chlamydia sp., Dictyostelium discoideum, Helicobacter pylori (Q9ZJ93), Mycobacterium tuberculosis, Pyrococcus abyssi (Q9UZ51), Pyrococcus horikoshii, Rickettsia prowazekii, Saccharolobus solfataricus, Treponema pallidum
Manually annotated by BRENDA team
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
Manually annotated by BRENDA team
Leduc, D.; Graziani, S.; Lipowski, G.; Marchand, C.; Le Marechal, P.; Liebl, U.; Myllykallio, H.
Functional evidence for active site location of tetrameric thymidylate synthase X at the interphase of three monomers
Proc. Natl. Acad. Sci. USA
101
7252-7257
2004
Helicobacter pylori
Manually annotated by BRENDA team
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
Manually annotated by BRENDA team
Escartin, F.; Skouloubris, S.; Liebl, U.; Myllykallio, H.
Flavin-dependent thymidylate synthase X limits chromosomal DNA replication
Proc. Natl. Acad. Sci. USA
105
9948-9952
2008
Helicobacter pylori, Paramecium bursaria Chlorella virus 1
Manually annotated by BRENDA team
Zhang, X.; Zhang, J.; Guo, G.; Mao, X.; Hu, Y.; Zou, Q.
Crystal structure of a flavin-dependent thymidylate synthase from Helicobacter pylori strain 26695
Protein Pept. Lett.
19
1225-1230
2012
Helicobacter pylori
Manually annotated by BRENDA team
Wang, K.; Wang, Q.; Chen, J.; Chen, L.; Jiang, H.; Shen, X.
Crystal structure and enzymatic characterization of thymidylate synthase X from Helicobacter pylori strain SS1
Protein Sci.
20
1398-1410
2011
Helicobacter pylori (Q5UVJ4), Helicobacter pylori, Helicobacter pylori SS1 (Q5UVJ4)
Manually annotated by BRENDA team
Skouloubris, S.; Djaout, K.; Lamarre, I.; Lambry, J.C.; Anger, K.; Briffotaux, J.; Liebl, U.; de Reuse, H.; Myllykallio, H.
Targeting of Helicobacter pylori thymidylate synthase ThyX by non-mitotoxic hydroxy-naphthoquinones
Open Biology
5
150015
2015
Helicobacter pylori (O26061), Helicobacter pylori, Helicobacter pylori 26695 (O26061)
Manually annotated by BRENDA team