Information on EC 1.3.98.1 - dihydroorotate dehydrogenase (fumarate)

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The expected taxonomic range for this enzyme is: Eukaryota, Bacteria, Archaea

EC NUMBER
COMMENTARY hide
1.3.98.1
-
RECOMMENDED NAME
GeneOntology No.
dihydroorotate dehydrogenase (fumarate)
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
(S)-dihydroorotate + fumarate = orotate + succinate
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
-
-
-
-
redox reaction
-
-
-
-
reduction
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
UMP biosynthesis II
-
-
pyrimidine metabolism
-
-
Pyrimidine metabolism
-
-
Metabolic pathways
-
-
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 hide
9029-03-2
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
patients with rheumatoid arthritis
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
enzyme type 1S
-
-
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
EATRO 110
-
-
Manually annotated by BRENDA team
strain Y
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
-
integration of a single copy of the yeast fumarate-dependent dihydroorotate dehydrogenase gene yDHODH into the genomes of strains D10attB, 3D7attB, Dd2attB, and HB3attB of Plasmodium falciparum. The yeast gene is equally expressed in all of the transgenic lines. All four yeast dihydroorotate dehydrogenase transgenic lines show strong resistance to atovaquone in standard short-term growth inhibition assays. During longer term growth with atovaquone, D10attB-yDHODH and 3D7attB-yDHODH parasites remain fully resistant, but Dd2attB-yDHODH and HB3attB-yDHODH parasites lose their tolerance to the drug after 3 to 4 days of exposure. No differences are found in growth responses among all of these strains to the Plasmodium-specific DHODH inhibitor DSM1 in either short- or long-term exposures. The ubiquinone analog decylubiquinone substantially reverses the atovaquone inhibition of Dd2attB-yDHODH and HB3attByDHODH transgenic parasites during extended growth
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(S)-dihydroorotate + acceptor
orotate + reduced acceptor
show the reaction diagram
(S)-dihydroorotate + ferricyanide
orotate + ferrocyanide
show the reaction diagram
(S)-dihydroorotate + fumarate
orotate + succinate
show the reaction diagram
(S)-dihydroorotate + O2
orotate + H2O2
show the reaction diagram
dihydroorotate + 2,3-dimethoxy-5-methyl-6-(3-methyl-2-butenyl)-1,4-benzoquinone
orotate + reduced 2,3-dimethoxy-5-methyl-6-(3-methyl-2-butenyl)-1,4-benzoquinone
show the reaction diagram
-
-
-
?
dihydroorotate + 2,6-dichloroindophenol
orotate + reduced 2,6-dichloroindophenol
show the reaction diagram
-
-
-
?
dihydroorotate + 2,6-dichlorophenolindophenol
orotate + ?
show the reaction diagram
-
-
-
-
?
dihydroorotate + 2,6-dichlorophenolindophenol
orotate + reduced 2,6-dichlorophenolindophenol
show the reaction diagram
dihydroorotate + acceptor
orotate + reduced acceptor
show the reaction diagram
dihydroorotate + ferricyanide
orotate + ferrocyanide
show the reaction diagram
-
-
-
-
?
dihydroorotate + fumarate
orotate + ?
show the reaction diagram
dihydroorotate + fumarate
orotate + reduced fumarate
show the reaction diagram
dihydroorotate + fumarate
orotate + succinate
show the reaction diagram
-
prefers fumarate over ubiquinone-1
-
-
?
dihydroorotate + O2
orotate + H2O2
show the reaction diagram
dihydroorotate + ubiquinone-1
orotate + ubiquinol-1
show the reaction diagram
-
-
-
-
?
dihydrooxonate + acceptor
oxonate + reduced acceptor
show the reaction diagram
L-dihydroorotate + 2,6-dichloroindophenol
orotate + ?
show the reaction diagram
-
-
-
-
?
L-dihydroorotate + 2,6-dichlorophenolindophenol
orotate + reduced 2,6-dichlorophenolindophenol
show the reaction diagram
-
-
-
-
?
L-dihydroorotate + CoQ
orotate + ?
show the reaction diagram
-
-
-
-
?
L-dihydroorotate + crotonate
orotate + butanoate
show the reaction diagram
-
-
-
-
?
L-dihydroorotate + fumarate
orotate + succinate
show the reaction diagram
L-dihydroorotate + mesaconate
orotate + 2-methylbutanedioate
show the reaction diagram
-
-
-
-
?
L-dihydroorotate + O2
orotate + H2O2
show the reaction diagram
-
-
-
-
?
orotate + ferricyanide
(S)-dihydroorotate + ferrocyanide
show the reaction diagram
-
-
-
-
?
S-dihydroorotate + 2,6-dichlorophenolindophenol
orotate + reduced 2,6-dichlorophenolindophenol
show the reaction diagram
-
-
-
-
?
S-dihydroorotate + fumarate
orotate + succinate
show the reaction diagram
-
100% activity
-
-
?
S-dihydroorotate + menadione
orotate + menadiol
show the reaction diagram
-
20% activity with menadione compared to fumarate
-
-
?
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
(S)-dihydroorotate + acceptor
orotate + reduced acceptor
show the reaction diagram
(S)-dihydroorotate + fumarate
orotate + succinate
show the reaction diagram
-
enzyme is responsible for biosynthesis of succinate and orotate
-
-
?
(S)-dihydroorotate + O2
orotate + H2O2
show the reaction diagram
L-dihydroorotate + fumarate
orotate + succinate
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
ferricyanide
-
-
flavin
ubiquinone-0
-
-
additional information
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
(NH4)2SO4
-
enhances enzymatic activity up to a concentration of 0.0014 mM
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,5-dihydrofuro[3,4-d]pyrimidine-2,4,7-trione
-
-
1-(1-naphthyl)-3-[2-(trifluoromethyl)phenyl]urea
-
50% inhibition at 0.0004 mM, wild type, 0.00034 mM, mutant R265A, above 0.2, mutant H185A
1-(2-methoxyphenyl)-3-(1-naphthyl)urea
-
50% inhibition at 0.00023 mM, wild type, 0.00024 mM, mutant R265A, 0.140, mutant H185A
1-(4-Chloro-phenyl)-1H-pyrazole-3,5-dicarboxylic acid 3-(4-methyl-benzylamide) 5-phenylamide
-
; minimum inhibitory concentration 0.032 mg/ml
1-(4-Chloro-phenyl)-1H-pyrazole-3,5-dicarboxylic acid 3-benzylamide 5-(isobutyl-amide)
-
; minimum inhibitory concentration above 0.064 mg/ml
1-(4-Chloro-phenyl)-1H-pyrazole-3,5-dicarboxylic acid 3-benzylamide 5-[(4-carbamoyl-phenyl)-amide]
-
; minimum inhibitory concentration 0.008 mg/ml
1-(4-Chloro-phenyl)-1H-pyrazole-3,5-dicarboxylic acid 5-phenylamide 3-[(pyridin-3-ylmethyl)-amide]
-
; minimum inhibitory concentration 0.00025 mg/ml
1-(4-Chloro-phenyl)-1H-pyrazole-3,5-dicarboxylic acid 5-[(4-piperidin-1-yl-phenyl)-amide] 3-[(pyridin-3-ylmethyl)-amide]
-
; minimum inhibitory concentration 0.002 mg/ml
1-(4-chlorophenyl)-3-quinolin-8-ylurea
-
50% inhibition at 0.00078 mM, wild type, 0.002 mM, mutant R265A, above 0.4, mutant H185A
1-(4-Methoxy-phenyl)-1H-pyrazole-3,5-dicarboxylic acid 3-(isobutyl-amide) 5-[[4-(pyrrolidine-1-carbonyl)-phenyl]-amide]
-
; minimum inhibitory concentration 0.064 mg/ml
1-(4-Methoxy-phenyl)-1H-pyrazole-3,5-dicarboxylic acid 3-benzhydryl-amide 5-benzylamide
-
; minimum inhibitory concentration above 0.032 mg/ml
1-(4-Methoxy-phenyl)-1H-pyrazole-3,5-dicarboxylic acid 3-benzylamide 5-[[4-(pyrrolidine-1-carbonyl)-phenyl]-amide]
-
; minimum inhibitory concentration 0.003 mg/ml
1-(4-Methoxy-phenyl)-1H-pyrazole-3,5-dicarboxylic acid 5-benzylamide 3-[[4-(pyrrolidine-1-carbonyl)-phenyl]-amide]
-
; minimum inhibitory concentration above 0.032 mg/ml
1-(4-Methoxy-phenyl)-1H-pyrazole-3,5-dicarboxylic acid 5-phenylamide 3-[(pyridin-3-ylmethyl)-amide]
-
; minimum inhibitory concentration 0.0005 mg/ml
1-(4-Methoxy-phenyl)-1H-pyrazole-3,5-dicarboxylic acid 5-[[4-(pyrrolidine-1-carbonyl)-phenyl]-amide] 3-[(tetrahydro-furan-2-yl)-amide]
-
; minimum inhibitory concentration above 0.032 mg/ml
2,3-dihydrotriazolo[4,5-d]pyrimidine-5,7-dione
-
-
2,6-dioxo-1,2,3,6-tetrahydropyrimidine-4-carboxylic acid
-
-
2-methyl-3-nitro-N-[3-(trifluoromethyl)phenyl]benzamide
-
50% inhibition at 0.00008 mM, wild type, 0.00037 mM, mutant R265A, 0.120, mutant H185A
2-methyl-N-1-naphthyl-3-nitrobenzamide
-
50% inhibition at 0.00008 mM, wild type, 0.00048 mM, mutant R265A, 0.180, mutant H185A
2-thioorotate
-
orotate analogue, 50% inhibition at 0.018 mM
3,4-dihydroxybenzoate
3,5-Dihydroxybenzoate
4,6-dioxo-1,4,5,6-tetrahydro-1,3,5-triazine-2-carboxylic acid
-
-
5-amino-2,6-dioxo-1,2,3,6-tetrahydropyrimidine-4-carboxylic acid
-
-
5-aminoorotate
-
orotate analogue, 50% inhibition at 1.6 mM
5-bromoorotate
-
orotate analogue, 50% inhibition at 0.016 mM
5-fluoro-2,6-dioxo-1,2,3,6-tetrahydropyrimidine-4-carboxylic acid
-
-
5-Fluoroorotate
-
orotate analogue, 50% inhibition at 0.055 mM
5-nitro-2,6-dioxo-1,2,3,6-tetrahydropyrimidine-4-carboxylic acid
-
-
5-nitroorotate
-
orotate analogue, 50% inhibition at 0.076 mM
5-[(dimethylamino)methyl]-2,6-dioxo-1,2,3,6-tetrahydropyrimidine-4-carboxylic acid
-
-
7,9-dihydro-3H-purine-2,6,8-trione
-
-
Barbiturate
-
dead-end inhibitor, competitive with respect to (S)-dihydroorotate
butyl 2,6-dioxo-1,2,3,6-tetrahydropyrimidine-4-carboxylate
-
-
leflunomide
-
cell line A20R resistant to leflunomide shows enhanced expression of enzyme to bypass the inhibitory effect, following long-time exposure, enzyme gene amplification can be assumed
Mercuric chloride
-
40% inhibition after 15 min at 0.000045 mM
methanol extract of brown algae Fucus evanescens
-
59% inhibition, noncompetitive
-
methanol extract of brown algae Pelvetia babingtonii
-
58% inhibition, noncompetitive
-
N-(2,4-dichlorophenyl)-2-naphthamide
-
50% inhibition at 0.00005 mM, wild type, 0.00008 mM, mutant R265A, above 0.05, mutant H185A
N-(2-fluorophenyl)naphthalene-2-carboxamide
-
50% inhibition at 0.00047 mM, wild type, 0.00030 mM, mutant R265A, 0.21, mutant H185A
N-(3,4-dichlorophenyl)-2-methyl-3-nitrobenzamide
-
50% inhibition at 0.00008 mM, wild type, 0.0028 mM, mutant R265A, 0.06, mutant H185A
N-(3,4-difluorophenyl)-2-methyl-3-nitrobenzamide
-
50% inhibition at 0.00026 mM, wild type, 0.00082 mM, mutant R265A, 0.25, mutant H185A
N-(3,5-dichlorophenyl)-2-methyl-3-nitrobenzamide
-
50% inhibition at 0.00002 mM, wild type, 0.0004 mM, mutant R265A, 0.13, mutant H185A
N-(3-bromophenyl)-2-methyl-3-nitrobenzamide
-
50% inhibition at 0.00006 mM, wild type, 0.0012 mM, mutant R265A, 0.140, mutant H185A
N-(3-chloro-4-fluorophenyl)-2-methyl-3-nitrobenzamide
-
50% inhibition at 0.0001 mM, wild type, 0.0005 mM, mutant R265A, 0.13, mutant H185A
N-(4-bromo-2-methylphenyl)-2-naphthamide
-
50% inhibition at 0.00005 mM, wild type, 0.00005 mM, mutant R265A, 0.57, mutant H185A
N3-benzyl-1-(4-chlorophenyl)-N5-phenyl-1H-pyrazole-3,5-dicarboxamide
-
; minimum inhibitory concentration 0.000125 mg/ml
Orotate
orotate methyl ester
-
orotate analogue, 50% inhibition at 0.71 mM
oxonate
-
crystallization data
oxonic acid
-
orotate analogue, 50% inhibition at 0.0099 mM
p-hydroxymercuribenzoate
-
98% inhibition after 5 min at 0.00227 mM
Salicylhydroxamic acid
-
weak inhibition, pH 9.0, 37C
suramin
-
weak inhibition, pH 9.0, 37C
Tiron
-
inhibits cytochrome c reduction and sulfite autoxidation
Uracil
-
orotate analogue, 50% inhibition at 2.5 mM
additional information
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-mercaptoethanol
-
0.175 mM restores inhibited enzyme after 3 min incubation to 90% of full activity
IPTG
-
induces expression
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00856 - 0.026
(S)-dihydroorotate
0.0131
2,3-dimethoxy-5-methyl-6-(3-methyl-2-butenyl)-1,4-benzoquinone
at saturating concentration of dihydroorotate
0.115
2,6-dichloroindophenol
-
in 50mM Tris/HCl, 150 mM KCl, 0.1% (v/v) Triton X-100, pH 8, at 30C
0.016 - 0.066
CoQ
0.0192 - 0.071
dihydroorotate
0.001 - 0.344
ferricyanide
0.03 - 0.4135
fumarate
0.0086 - 0.123
L-dihydroorotate
0.001 - 0.5
S-dihydroorotate
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
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
39
dihydrooxonate
Saccharomyces cerevisiae
-
with ferricyanide
157
ferricyanide
Lactobacillus delbrueckii subsp. bulgaricus
-
-
8.5
fumarate
Trypanosoma brucei
-
apparent value
1.7 - 7.8
L-dihydroorotate
2.7 - 231
S-dihydroorotate
additional information
2,3-dimethoxy-5-methyl-6-(3-methyl-2-butenyl)-1,4-benzoquinone
Plasmodium falciparum
Q08210
at saturating concentration of dihydroorotate
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.000032 - 0.05
1-(4-Chloro-phenyl)-1H-pyrazole-3,5-dicarboxylic acid 3-(4-methyl-benzylamide) 5-phenylamide
0.000091 - 0.05
1-(4-Chloro-phenyl)-1H-pyrazole-3,5-dicarboxylic acid 3-benzylamide 5-(isobutyl-amide)
0.000006 - 0.05
1-(4-Chloro-phenyl)-1H-pyrazole-3,5-dicarboxylic acid 3-benzylamide 5-[(4-carbamoyl-phenyl)-amide]
0.000004 - 0.05
1-(4-Chloro-phenyl)-1H-pyrazole-3,5-dicarboxylic acid 5-phenylamide 3-[(pyridin-3-ylmethyl)-amide]
0.000008 - 0.05
1-(4-Chloro-phenyl)-1H-pyrazole-3,5-dicarboxylic acid 5-[(4-piperidin-1-yl-phenyl)-amide] 3-[(pyridin-3-ylmethyl)-amide]
0.000138 - 0.05
1-(4-Methoxy-phenyl)-1H-pyrazole-3,5-dicarboxylic acid 3-(isobutyl-amide) 5-[[4-(pyrrolidine-1-carbonyl)-phenyl]-amide]
0.0024 - 0.05
1-(4-Methoxy-phenyl)-1H-pyrazole-3,5-dicarboxylic acid 3-benzhydryl-amide 5-benzylamide
0.000026 - 0.05
1-(4-Methoxy-phenyl)-1H-pyrazole-3,5-dicarboxylic acid 3-benzylamide 5-[[4-(pyrrolidine-1-carbonyl)-phenyl]-amide]
0.0015 - 0.05
1-(4-Methoxy-phenyl)-1H-pyrazole-3,5-dicarboxylic acid 5-benzylamide 3-[[4-(pyrrolidine-1-carbonyl)-phenyl]-amide]
0.000012 - 0.05
1-(4-Methoxy-phenyl)-1H-pyrazole-3,5-dicarboxylic acid 5-phenylamide 3-[(pyridin-3-ylmethyl)-amide]
0.000331 - 0.05
1-(4-Methoxy-phenyl)-1H-pyrazole-3,5-dicarboxylic acid 5-[[4-(pyrrolidine-1-carbonyl)-phenyl]-amide] 3-[(tetrahydro-furan-2-yl)-amide]
0.000004 - 0.05
N3-benzyl-1-(4-chlorophenyl)-N5-phenyl-1H-pyrazole-3,5-dicarboxamide
0.0045 - 0.1
Orotate
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.
-
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0077
L-orotate
Saccharomyces cerevisiae
-
in 50mM Tris/HCl, 150 mM KCl, 0.1% (v/v) Triton X-100, pH 8, at 30C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.0011
-
cytosolic fraction, native enzyme
0.06
-
using crotonate as electron acceptor
0.09
-
pH 8.0, 25C, mutant N67A, substrate O2
0.11
-
pH 8.0, 25C, mutant N67A, substrate dichlorophenolindophenol
0.14
-
pH 8.0, 25C, mutant N193A, substrate fumarate
0.15
-
pH 8.0, 25C, mutant N193A, substrate dichlorophenolindophenol; pH 8.0, 25C, mutant P56A, substrate O2
0.17
-
pH 8.0, 25C, mutant N193A, substrate O2
0.3
-
pH 8.0, 25C, mutant N67A, substrate fumarate
0.47
-
pH 8.0, 25C, mutant P56A, substrate dichlorophenolindophenol
0.48
-
pH 8.0, 25C, mutant P56A, substrate fumarate
0.75
-
pH 8.0, 25C, mutant N127A, substrate dichlorophenolindophenol
0.94
-
using mesaconate as electron acceptor
0.987
-
using 0.02 mM ubiquinone-1 as acceptor, at 25C
1
-
pH 8.0, 25C, mutant N127A, substrate O2
1.2
-
pH 8.0, 25C, mutant N127A, substrate fumarate
1.4
-
at 37C, 5 mM sodium diphosphate buffer
1.6
-
pH 8.0, 25C, mutant K136E, substrate O2
1.61
-
purified native enzyme, electron acceptor fumarate
1.8
-
pH 8.0, 25C, mutant S129A, substrate O2
1.81
-
using O2 as electron acceptor
1.9
-
pH 8.0, 25C, mutant K213E, substrate O2
2
-
pH 8.0, 25C, wild-type, substrate O2
2.3
-
pH 8.0, 25C, mutant P131A, substrate O2
2.4
-
pH 8.0, 25C, mutant K213E, substrate dichlorophenolindophenol
2.8
-
pH 8.0, 25C, mutant S129A, substrate fumarate
2.95
-
purified recombinant enzyme, electron acceptor fumarate
3.3
-
pH 8.0, 25C, mutant R50E, substrate O2; pH 8.0, 25C, mutant R57A, substrate O2
3.318
-
using 0.5 mM fumarate as acceptor, at 25C
3.7
-
pH 8.0, 25C, mutant P131A, substrate fumarate
3.99
-
pH 9.0, 37C
4
-
pH 8.0, 25C, mutant P131A, substrate dichlorophenolindophenol
5.1
-
using fumarate as electron acceptor
7.2
-
pH 8.0, 25C, mutant K213E, substrate fumarate; pH 8.0, 25C, mutant R50E, substrate dichlorophenolindophenol
11.7
-
pH 8.0, 25C, mutant S129A, substrate dichlorophenolindophenol
13
-
pH 8.0, 25C, mutant K136E, substrate dichlorophenolindophenol
13.6
-
pH 8.0, 25C, wild-type, substrate dichlorophenolindophenol
15.8
-
pH 8.0, 25C, wild-type, substrate fumarate
18.1
-
pH 8.0, 25C, mutant K136E, substrate fumarate
19
-
pH 8.0, 25C, mutant R50E, substrate fumarate
27
-
pH 8.0, 25C, mutant R57A, substrate fumarate
65.7
-
pH 8.0, 25C, mutant R57A, substrate dichlorophenolindophenol
117.5
-
with ferricyanide as acceptor
3948
-
pH 8.5, 70C
151000
-
cosubstrate fumarate, pH 7.5
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5
-
assay at
7.7
-
with ferricyanide as electron acceptor
9.4
-
5 mM sodium diphosphate buffer
9.5
-
maximum, 100 mM sodium diphosphate
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 11
-
best in sodium phosphate and sodium diphosphate buffer, not active below pH 6.0, 60% of activity maximum at pH 11.0
6 - 10.5
-
assay in this range
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.5
calculated from amino acid sequence
6
-
isoelecric focusing
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
cell line A20R generated by gradually exposing A20N cells to increasing concentrations of enzyme inhibitor leflunomide
Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
UNIPROT
Lactococcus lactis subsp. cremoris (strain MG1363)
Lactococcus lactis subsp. cremoris (strain MG1363)
Lactococcus lactis subsp. cremoris (strain MG1363)
Lactococcus lactis subsp. cremoris (strain MG1363)
Lactococcus lactis subsp. cremoris (strain MG1363)
Lactococcus lactis subsp. cremoris (strain MG1363)
Lactococcus lactis subsp. cremoris (strain MG1363)
Lactococcus lactis subsp. cremoris (strain MG1363)
Lactococcus lactis subsp. cremoris (strain MG1363)
Lactococcus lactis subsp. cremoris (strain MG1363)
Lactococcus lactis subsp. cremoris (strain MG1363)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
34100
DHOD3, sequence analysis
34200
DHOD1 and DHOD2, sequence analysis
37000
recombinant DHOD1, DHOD2 and DHOD3 with an N-terminal His6-tag, affinity chromatography
50000
gel filtration
53000
-
gel filtration, crude fraction
61000
-
SDS-PAGE
62000
-
gel filtration
63200
-
gel filtration
63300
-
gel filtration
66000
-
gel filtration
72000
-
sucrose-density-gradient centrifugation
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
x * 65100, deduced from gene sequence, x * 66200, SDS-PAGE
heteromer
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x * catalytic subunit, 31100, + x * electron acceptor subunit, 23600, SDS-PAGE
homodimer
monomer
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crystallization data
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
in complex with product orotate
-
enzyme in complex with orotate, crystal structure analysis, overview
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; dihydroorotate dehydrogenase A, complexed with orotate
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; hanging drop vapor diffusion technique, 30% polyethylene glycol, 0.2 M sodium acetate, 0.1 M Tris-HCl, pH 8.5, monoclinic crystals, space group P21
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hanging drop vapour diffusion method, with 30% PEG 6000, 1 mM dithiothreitol, 0.2 M sodium acetate, and 0.1 M Tris-HCl at pH 8.5
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wild-type enzyme and mutants N67A, K213E, P56A, R57A, K136E
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apo-enzyme and in complex with orotate and with fumarate, to 2.0 A, 2.5 A and 1.9 A resolution, respectively. Both orotate and fumarate bind to the same active site and exploit similar interactions, consistent with a ping-pong mechanism. Rearrangements in the conformation of the catalytic loop have direct influence on the dimeric interface
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by the vapour-diffusion technique using lithium sulfate as the precipitating agent, to better than 2.0 A resolution, crystals belong to space group P61, presence of two molecules in the asymmetric unit; crystallized by the vapour-diffusion technique using sitting and hanging drops (lithium sulfate as the precipitating agent). The crystals belong to space group P6(1), with unit-cell parameters a = 143.7, c = 69.8 A. X-ray diffraction data are collected to 2.0 A resolution using an in-house rotating-anode generator
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by sitting-drop vapor-diffusion technique, to 2.4 A resolution
to 2.4 A resolution, space group C2221. Class 1A family enzyme, the monomer folds as a alpha/beta barrel consisting of a core of central eight parallel beta-strands surrounded by a ring of eight alpha-helices. The asymmetric unit of the crystal structure contains four monomers that are arranged as two distinct homodimers that are nearly perpendicular to each other, i.e. the tetramer has the shape of the letter L
crystals of the TcDHODorotate complex are grown at 4C 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 -173C 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; 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
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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
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4
-
10% loss of activity per day
42
-
pH 9.0, enzyme not indefinitely stable at this temperature
45
-
20 min, loss of half of the activity
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
20% loss of activity after 1 h exposure to room light
-
Triton X-100 stabilizes
-
unstable to freezing and thawing
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ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY hide
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
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, 4 months
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-20C, 50% glycerol
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-20C, 50% glycerol, several months, without loss of activity
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-20C, 50% glycerol, stable for several months
-
-20C, progressive enzyme decay
-
-78C, indefinitely stable
-
0C, 1 week
-
4C, 10% loss of activity per day, full activity restored by addition of 0.1 mM 2-mercaptoethanol or dithiothreitol
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4C, pH 7.8, 0.05 M sodium phosphate buffer, 0.1% Triton X-100
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4C, purified enzyme, stable up to two years
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
; 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
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; Orange-A column, reverse-affinity step, Mono Q column
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; streptomycin precipitation, ion-exchange, hydroxylapatite
-
acid treatment, DE-52-, Matrex Orange A-, Matrex Blue A column chromatography
-
ammonium sulfate precipitation and Q-Sepharose column chromatography
-
by ion-exchange chromatography
-
centrifugation after cell disruption, separation from the NADP-linked dihydroorotic dehydrogenase
-
ion-exchange, gel filtration, ammonium sulfate precipitation; preparative gel electrophoresis
-
Ni2+-NTA agarose chromatography, Q-Sepharose column chromatography, and Superdex 200 gel filtration
-
preparative gel electrophoresis
-
recombinant DHOD1, DHOD2 and DHOD3 with an N-terminal His6-tag, by affinity chromatography
recombinant His-tagged enzyme by nickel affinity chromatography
-
recombinant; to homogeneity by ion-exchange, hydrophobic interaction and gel filtration
-
soniciation and gel filtration
streptomycin sulfate and ammonium sulfate precipitation, DEAE-Sephacel-, affinity chromatography
-
the enzyme is fused with aspartate carbamoyltransferase as precursor to mature DHOD
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to homogeneity by a single affinity chromatography step
-
to homogeneity by a single nickel affinity chromatography step
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
; expression of His-tagged enzyme
-
expressed as His6-tag fusion protein
-
expressed in Escherichia coli
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expressed in Escherichia coli strain BL21-RP
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expressed in Escherichia coli strain S6645
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expressed in SO6645 cells
-
expression in Escherichia coli
expression in Escherichia coli strain BL21(DE3) as His-tagged enzyme
-
expression in Escherichia coli; expression in Escherichia coli; expression in Escherichia coli; 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
expression in Plasmodium falciparum
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into vector pET-28a and overexpressed in Escherichia coli strain BL21(DE3) as a histidine-tag fusion protein
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into vector pRSET and expressed in either freshly transformed or glycerol stocked Escherichia coli BL21-CodonPlus (DE3)-RIL
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
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transformation in Escherichia coli for detection of pyrimidine prototrophic colonies with a resistance of ampicillin
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
S175A
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low activity with dihydroorotate, increasing activity above pH 8.0 with dihydrooxonate
Q7L
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single nucleotide polymorphism due to missense polymorphism 19C>A, leading to amino acid substitution in the cationic N-terminal region of the polypeptide
E206/K296E
-
conversion of intermolecular salt bridge, mutant is fully active in concentrated solutions and dissociates into monomers upon dilution like wild-type enzyme
E206A
-
disturbance of intermolecular salt bridge, mutant retains almost complete activity
E206K
-
disturbance of intermolecular salt bridge, mutant activity is severely impaired
K136E
-
little changes in activity
K213E
-
decrease in activity against dichlorophenolindophenol
K296A
-
disturbance of intermolecular salt bridge, mutant retains almost complete activity
K296E
-
disturbance of intermolecular salt bridge, mutant activity is severely impaired
L71F
-
reaction is somehow slower than for wild-type, binding of dihydroorotate is much tighter than with wild-type
L71F/C130S/V133T
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addition of the two residues comprising the conserved proton-transfer network of Class 2 dihydroorotate dehydrogenase from Escherichia coli to the C130S Class 1A enzyme of Lactococcus lacits. Mutation does not did not restore the function of the active site base or rapid flavin reduction. Kd for dihydroorotate is about three times tighter than the wild-type
L71F/V133T
-
reaction is drastically slower forwith wild-type, Kd for dihydroorotate is iabout eight-fold tighter than the wild-type
N127A
-
reduced activity
N193A
-
drastically reduced activiy
N67A
-
drastically reduced activiy
P131A
-
decrease in activtiy against fumarate
P56A
-
drastically reduced activiy
R50E
-
little changes in activity
R57A
-
increase in activity
S129A
-
decrease in activtiy against fumarate
V133T
-
reaction is somehow slower than for wild-type, binding of dihydroorotate is much weaker than with wild-type
H185A
-
4fold increase in KM-value of CoQD, 50% increase in KM-value of L-dihydroorotate
R265A
-
2fold increase in KM-value of CoQD, 15% increase in KM-value of L-dihydroorotate
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
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
Show AA Sequence (935 entries)
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