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Information on EC 1.3.98.1 - dihydroorotate dehydrogenase (fumarate) and Organism(s) Trypanosoma cruzi and UniProt Accession Q4D3W2
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1.3.98.1 dihydroorotate dehydrogenase (fumarate)IUBMB Comments
Binds FMN. The reaction, which takes place in the cytosol, is the only redox reaction in the de novo biosynthesis of pyrimidine nucleotides. Molecular oxygen can replace fumarate in vitro. Other class 1 dihydroorotate dehydrogenases use either NAD+ (EC 1.3.1.14) or NADP+ (EC 1.3.1.15) as electron acceptor. The membrane bound class 2 dihydroorotate dehydrogenase (EC 1.3.5.2) uses quinone as electron acceptor.
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Word Map
- 1.3.98.1
- pyrimidine
- leflunomide
- brequinar
- uridine
- plasmodium
- falciparum
- ubiquinone
- teriflunomide
-
dhods
-
1.3.3.1
- ump
-
dihydro-orotate
- triazolopyrimidine
- atovaquone
-
1.3.99.11
- dysostosis
- analysis
- medicine
-
acrofacial
The taxonomic range for the selected organisms is: Trypanosoma cruzi
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Synonyms
dhodh, dhodehase, dhoda, lmdhodh, dihydroorotate oxidase, tcdhod, (dho) dehydrogenase, class 1a dhod, class 1a dhodh, dhod1, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
(DHO) dehydrogenase
-
-
-
-
4,5-L-dihydroorotate:oxygen oxidoreductase
-
-
-
-
DHOdehase
-
-
-
-
Dihydroorotate oxidase
-
-
-
-
oxidase, dihydroorotate
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-
-
-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
(S)-dihydroorotate + fumarate = orotate + succinate
ping-pong bi-bi mechanism
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
redox reaction
-
-
-
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oxidation
-
-
-
-
reduction
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-
-
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SYSTEMATIC NAME
IUBMB Comments
(S)-dihydroorotate:fumarate oxidoreductase
Binds FMN. The reaction, which takes place in the cytosol, is the only redox reaction in the de novo biosynthesis of pyrimidine nucleotides. Molecular oxygen can replace fumarate in vitro. Other class 1 dihydroorotate dehydrogenases use either NAD+ (EC 1.3.1.14) or NADP+ (EC 1.3.1.15) as electron acceptor. The membrane bound class 2 dihydroorotate dehydrogenase (EC 1.3.5.2) uses quinone as electron acceptor.
CAS REGISTRY NUMBER
COMMENTARY
9029-03-2
-
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
dihydroorotate + 2,6-dichlorophenolindophenol
orotate + reduced 2,6-dichlorophenolindophenol
-
-
-
-
?
dihydroorotate + fumarate
orotate + reduced fumarate
-
-
-
?
additional information
?
-
-
enzyme has methylviologen-fumarate reductase activity
-
-
?
(S)-dihydroorotate + fumarate
orotate + succinate
-
enzyme is responsible for biosynthesis of succinate and orotate
-
-
?
L-dihydroorotate + fumarate
orotate + succinate
-
DHODH catalyzes a coupled redox reaction in which dihydroorotate is oxidized to orotate and fumarate is reduced to succinate
-
-
?
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
(S)-dihydroorotate + fumarate
orotate + succinate
-
enzyme is responsible for biosynthesis of succinate and orotate
-
-
?
L-dihydroorotate + fumarate
orotate + succinate
-
DHODH catalyzes a coupled redox reaction in which dihydroorotate is oxidized to orotate and fumarate is reduced to succinate
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
-
oxidized 2,6-dichlorophenol-indophenol, ferricyanide, or 1-ubiquinone can act as artificial electron acceptors
-
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
Barbiturate
-
dead-end inhibitor, competitive with respect to (S)-dihydroorotate
methanol extract of brown algae Fucus evanescens
-
59% inhibition, noncompetitive
-
methanol extract of brown algae Pelvetia babingtonii
-
58% inhibition, noncompetitive
-
Orotate
competitively inhibits all three DHOD enzymes to a comparable level
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.0086
L-dihydroorotate
-
pH not specified in the publication, temperature not specified in the publication
additional information
additional information
-
10% v/v dimethyl sulfoxide and 0.5% v/vTriton X-100, which seem to facilitate the substrate binding process with a small decrease in Km, enzyme-substrate complex, pseudo-first-order kinetics, thermodynamics, overview
-
0.00856
(S)-dihydroorotate
-
assay based on isothermal titration calorimetry, pH 8.0, 25°C
0.12
fumarate
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pH not specified in the publication, temperature not specified in the publication
0.12
fumarate
-
assay based on isothermal titration calorimetry, pH 8.0, 25°C
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
additional information
-
Ki-values are 0.0353 and 0.0103 mg per ml for methanol extract of Fucus evanescens and Pelvetia babingtonii, resp.
-
0.0156
Orotate
inhibition of DHOD2 at 25°C, in the presence of 1 mM fumarate
0.0205
Orotate
inhibition of DHOD2 at 25°C, in the presence of 1 mM dihydroorotate
0.0424
Orotate
inhibition of DHOD3 at 37°C, in the presence of 1 mM dihydroorotate
0.049
Orotate
inhibition of DHOD3 at 37°C, in the presence of 1 mM fumarate
0.0573
Orotate
inhibition of DHOD1 at 37°C, in the presence of 1 mM dihydroorotate
0.0622
Orotate
inhibition of DHOD1 at 37°C, in the presence of 1 mM fumarate
0.0714
Orotate
inhibition of DHOD2 at 37°C, in the presence of 1 mM dihydroorotate
0.0773
Orotate
inhibition of DHOD2 at 37°C, in the presence of 1 mM fumarate
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
34000
37000
recombinant DHOD1, DHOD2 and DHOD3 with an N-terminal His6-tag, affinity chromatography
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dimer
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
DHODorotate complex by the sitting-drop vapour-diffusion technique using polyethylene glycol 3350 as a precipitant, to better than 1.8 A resolution, crystals belong to the orthorhombic space group P212121, with unit-cell parameters a = 67.87, b = 71.89, c = 123.27 A
in ligand-free form and in complexes with inhibitor oxonate, physiological substrates and products of the first and second half-reactions. Ligands bind to the same active site of enzyme, consistent with one-site ping-pong Bi-Bi mechanism. The binding of ligands does not cause any significant structural changes, and both reduced and oxidized FMN cofactors are in planar conformation. Resiude C130 is well located for abstracting a proton from dihydroorotate C5 and transferring it to outside water molecules. The bound fumarate is in a twisted conformation, which induces partial charge separation. The thermodynamically favorable reduction of fumarate with reduced FMN seems to proceed in the way that its C2 accepts a proton from C130 and C3 a hydride or a hydride equivalent from reduced FMN N5
crystals of the TcDHODorotate complex are grown at 4°C by the sitting-drop vapour-diffusion technique using polyethylene glycol 3350 as a precipitant. The crystals diffract to better than 1.8 A ° resolution using synchrotron radiation (lambda = 0.900 A). X-ray diffraction data are collected at -173°C and processed to 1.9 A ° resolution with 98.2% completeness. The TcDHOD crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 67.87, b = 71.89, c = 123.27 A
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
-
DHOD-knockout Trypanosoma cruzi do not express the enzyme protein and can not survive even in the presence of pyrimidine nucleosides, suggesting a vital role of fumarate reductase activity in the regulation of cellular redox balance
ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dimethyl sulfoxide
-
presence of dimethyl sulfoxide at 10%, v/v and Triton X-100 at 0.5%, v/v during the assay seems to facilitate the substrate binding process with a small decrease in KM value
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
to homogeneity by ion-exchange, hydrophobic interaction and gel filtration
native enzyme from the cytosolic fraction by gel filtration and immunoaffinity chromatography, recombinant His-tagged enzyme by nickel affinity chromatography to homogeneity and subsequent clevage of the tag by thrombin
-
recombinant DHOD1, DHOD2 and DHOD3 with an N-terminal His6-tag, by affinity chromatography
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
ligated into the pZErO-2 vector and transformation of the Escherichia coli strain TOP 10, inserted into the pET3a expression vector and transformation of Escherichia coli BL21
genes subcloned int the EcoRI site of pUC18, PCR products cloned in vector pET100/D-TOPO, recombinant DHOD1 and DHOD2 expressed in Escherichia coli BL21-CodonPlus (DE3)-RP
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
analysis
-
protocol to measure enzyme kinetic parameters based on isothermal titration calorimetry. Presence of dimethyl sulfoxide at 10%, v/v and Triton X-100 at 0.5%, v/v seems to facilitate the substrate binding process with a small decrease in KM value
medicine
additional information
-
the cytosolic DHOD gene acquired may have contributed to adaptation to anaerobiosis in the kinetoplastid lineage and further contributes to the subsequent establishment of parasitism in a trypanosomatid ancestor
medicine
-
enzyme is inhibited by 59% and 58% by methanol extracts of the brown algae Fucus evanescens and Pelvetia babingtonii, resp., the extracts decrease significantly the infection rate of HeLa host cells and the average parasite number per infected cell
medicine
despite their genetic variations, kinetic properties of the three DHODs are conserved, these findings facilitate further exploitation of Trypanosoma cruzi DHOD inhibitors, as chemotherapeutic agents against Chagas' disease
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Takashima, E.; Inaoka, D.K.; Osanai, A.; Nara, T.; Odaka, M.; Aoki, T.; Inaka, K.; Harada, S.; Kita, K.
Characterization of the dihydroorotate dehydrogenase as a soluble fumarate reductase in Trypanosoma cruzi
Mol. Biochem. Parasitol.
122
189-200
2002
Trypanosoma cruzi
Nara, T.; Kamei, Y.; Tsubouchi, A.; Annoura, T.; Hirota, K.; Iizumi, K.; Dohmoto, Y.; Ono, T.; Aoki, T.
Inhibitory action of marine algae extracts on the Trypanosoma cruzi dihydroorotate dehydrogenase activity and on the protozoan growth in mammalian cells
Parasitol. Int.
54
59-64
2005
Trypanosoma cruzi
Inaoka, D.K.; Takashima, E.; Osanai, A.; Shimizu, H.; Nara, T.; Aoki, T.; Harada, S.; Kita, K.
Expression, purification and crystallization of Trypanosoma cruzi dihydroorotate dehydrogenase complexed with orotate
Acta Crystallogr. Sect. F
61
875-878
2005
Trypanosoma cruzi, Trypanosoma cruzi (Q4D3W2)
Annoura, T.; Nara, T.; Makiuchi, T.; Hashimoto, T.; Aoki, T.
The origin of dihydroorotate dehydrogenase genes of kinetoplastids, with special reference to their biological significance and adaptation to anaerobic, parasitic conditions
J. Mol. Evol.
60
113-127
2005
Euglena gracilis, Euglena gracilis (Q75XR0), Neobodo saliens, Neobodo saliens (Q6F4D1), Parabodo caudatus (Q6F4D0), Trypanosoma cruzi
Sariego, I.; Annoura, T.; Nara, T.; Hashimoto, M.; Tsubouchi, A.; Iizumi, K.; Makiuchi, T.; Murata, E.; Kita, K.; Aoki, T.
Genetic diversity and kinetic properties of Trypanosoma cruzi dihydroorotate dehydrogenase isoforms
Parasitol. Int.
55
11-16
2006
Trypanosoma cruzi, Trypanosoma cruzi (Q4D3W2), Trypanosoma cruzi (Q4DEJ0), Trypanosoma cruzi (Q4DGV2)
Pinheiro, M.P.; Iulek, J.; Cristina Nonato, M.
Crystal structure of Trypanosoma cruzi dihydroorotate dehydrogenase from Y strain
Biochem. Biophys. Res. Commun.
369
812-817
2008
Trypanosoma cruzi, Trypanosoma cruzi Y
Inaoka, D.; Sakamoto, K.; Shimizu, H.; Shiba, T.; Kurisu, G.; Nara, T.; Aoki, T.; Kita, K.; Harada, S.
Structures of Trypanosoma cruzi dihydroorotate dehydrogenase complexed with substrates and products: Atomic resolution insights into mechanisms of dihydroorotate oxidation and fumarate reduction
Biochemistry
47
10881-10891
2008
Trypanosoma cruzi (Q4D3W2), Trypanosoma cruzi
Cheleski, J.; Wiggers, H.J.; Citadini, A.P.; da Costa Filho, A.J.; Nonato, M.C.; Montanari, C.A.
Kinetic mechanism and catalysis of Trypanosoma cruzi dihydroorotate dehydrogenase enzyme evaluated by isothermal titration calorimetry
Anal. Biochem.
399
13-22
2010
Trypanosoma cruzi
Cheleski, J.; Rocha, J.R.; Pinheiro, M.P.; Wiggers, H.J.; da Silva, A.B.; Nonato, M.C.; Montanari, C.A.
Novel insights for dihydroorotate dehydrogenase class 1A inhibitors discovery
Eur. J. Med. Chem.
45
5899-5909
2010
Trypanosoma cruzi (Q4D3W2), Trypanosoma cruzi
Silva, N.d.e..F.; Lameira, J.; Alves, C.N.; Marti, S.
Computational study of the mechanism of half-reactions in class 1A dihydroorotate dehydrogenase from Trypanosoma cruzi
Phys. Chem. Chem. Phys.
15
18863-18871
2013
Trypanosoma cruzi (Q4D3W2), Trypanosoma cruzi, Trypanosoma cruzi CL Brener (Q4D3W2)
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