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Information on EC 1.3.1.14 - dihydroorotate dehydrogenase (NAD+)
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1.3.1.14 dihydroorotate dehydrogenase (NAD+)IUBMB Comments
Binds FMN, FAD and a [2Fe-2S] cluster. The enzyme consists of two subunits, an FMN binding catalytic subunit and a FAD and iron-sulfur binding electron transfer subunit . The reaction, which takes place in the cytosol, is the only redox reaction in the de-novo biosynthesis of pyrimidine nucleotides. Other class 1 dihydroorotate dehydrogenases use either fumarate (EC 1.3.98.1) 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
- 1.3.1.14
- pyrimidine
- flavin
- brequinar
- medicine
- l-dihydroorotate
- fmn
- lactis
-
o2-sensitive
- mononucleotide
-
photons
- bifidobacterium
- bifidum
- berghei
- ubiquinone
-
carbamyl
-
half-reaction
- nutrition
- pharmacology
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1.3.3.1
The expected taxonomic range for this enzyme is: Bacteria, Archaea, Eukaryota
Synonyms
dhodase, dho dehydrogenase, b-type dihydroorotate dehydrogenase, orotate reductase, dihydro-orotic dehydrogenase, 
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SYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
DHO dehydrogenase
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DHODase
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-
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dihydro-orotic dehydrogenase
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-
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dihydroorotate dehydrogenase
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-
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dihydrorotate dehydrogenase B
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-
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L-5,6-dihydro-orotate:NAD oxidoreductase
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-
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orotate reductase
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-
-
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reductase, orotate
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-
-
-
additional information
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
(S)-dihydroorotate + NAD+ = orotate + NADH + H+
electron transfer mechanism
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(S)-dihydroorotate + NAD+ = orotate + NADH + H+
trans-dehydrogenation mechanism
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(S)-dihydroorotate + NAD+ = orotate + NADH + H+
ping-pong steady-state kinetic mechanism
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(S)-dihydroorotate + NAD+ = orotate + NADH + H+
ping-pong steady-state kinetic mechanism
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(S)-dihydroorotate + NAD+ = orotate + NADH + H+
PyrDI contains the dihydroorotate-binding site, but PyrDII is required for full activity in vivo. Holoenzyme joins a NAD+-reductase activity
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(S)-dihydroorotate + NAD+ = orotate + NADH + H+
detailed thermodynamic analysis, midpoint reduction potential of 2Fe-2S center -212 mV, midpoint reduction potential of FMN -298 mV
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(S)-dihydroorotate + NAD+ = orotate + NADH + H+
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-
-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dehydrogenation
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oxidation
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redox reaction
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reduction
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-
-
-
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PATHWAY SOURCE
PATHWAYS
BRENDA
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SYSTEMATIC NAME
IUBMB Comments
(S)-dihydroorotate:NAD+ oxidoreductase
Binds FMN, FAD and a [2Fe-2S] cluster. The enzyme consists of two subunits, an FMN binding catalytic subunit and a FAD and iron-sulfur binding electron transfer subunit [4]. The reaction, which takes place in the cytosol, is the only redox reaction in the de-novo biosynthesis of pyrimidine nucleotides. Other class 1 dihydroorotate dehydrogenases use either fumarate (EC 1.3.98.1) 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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CAS REGISTRY NUMBER
COMMENTARY
37255-26-8
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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
(S)-dihydroorotate + 2,6-dichlorophenolindophenol
orotate + reduced 2,6-dichlorophenolindophenol
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-
-
?
5-fluorodihydroorotate + NAD+
5-fluoroorotate + NADH + H+
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more active substrate for reverse reaction than orotate
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-
r
(S)-dihydroorotate + acceptor
orotate + reduced acceptor
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different specific activities with potassium ferricyanide, O2, fumarate and NAD+ as electron acceptors for PyrDI and the holoenzyme
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?
(S)-dihydroorotate + acceptor
orotate + reduced acceptor
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fourth step in UMP-biosynthesis
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?
(S)-dihydroorotate + NAD+
orotate + NADH + H+
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NAD+ as ultimate electron acceptor
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?
(S)-dihydroorotate + NAD+
orotate + NADH + H+
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NAD+ as ultimate electron acceptor
-
?
(S)-dihydroorotate + NAD+
orotate + NADH + H+
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NAD+ as ultimate electron acceptor
-
?
(S)-dihydroorotate + NAD+
orotate + NADH + H+
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NAD+ as ultimate electron acceptor
-
?
(S)-dihydroorotate + NAD+
orotate + NADH + H+
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mechanism and pH-dependence of reaction
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-
r
(S)-dihydroorotate + NAD+
orotate + NADH + H+
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anti-elimination of hydrogen from (S)-dihydroorotate
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r
(S)-dihydroorotate + NAD+
orotate + NADH + H+
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equilibrium favours direction of orotate reduction
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?
(S)-dihydroorotate + NAD+
orotate + NADH + H+
-
equilibrium favours direction of orotate reduction
-
r
(S)-dihydroorotate + NAD+
orotate + NADH + H+
-
equilibrium favours direction of orotate reduction
-
r
(S)-dihydroorotate + NAD+
orotate + NADH + H+
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equilibrium favours direction of orotate reduction
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r
(S)-dihydroorotate + NAD+
orotate + NADH + H+
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equilibrium favours direction of orotate reduction
orotate is identical with 4-carboxyuracil
r
(S)-dihydroorotate + NAD+
orotate + NADH + H+
-
NAD+ as ultimate electron acceptor
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?, r
(S)-dihydroorotate + NAD+
orotate + NADH + H+
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NAD+ as ultimate electron acceptor
-
r
(S)-dihydroorotate + NAD+
orotate + NADH + H+
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substrate is oxidized by NAD+ and oxygen
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r
(S)-dihydroorotate + NAD+
orotate + NADH + H+
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substrate is oxidized by NAD+ and oxygen
lipoic acid is no substitute for orotate
?
(S)-dihydroorotate + NAD+
orotate + NADH + H+
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mechanism of dehydrogenation, mechanism of electron transfer: L-dihydroorotate transfers a pair of electrons to FMN via iron-sulfur cluster via FAD to NAD+
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?, r
(S)-dihydroorotate + NAD+
orotate + NADH + H+
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mechanism of dehydrogenation, mechanism of electron transfer: L-dihydroorotate transfers a pair of electrons to FMN via iron-sulfur cluster via FAD to NAD+
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r
(S)-dihydroorotate + NAD+
orotate + NADH + H+
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NAD+ as ultimate electron acceptor
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?, r
(S)-dihydroorotate + NAD+
orotate + NADH + H+
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NAD+ as ultimate electron acceptor
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r
(S)-dihydroorotate + NAD+
orotate + NADH + H+
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at higher pH values reaction favours direction of dihydroorotate oxidation, whereas at lower pH values direction of orotate reduction is favoured
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r
dihydroorotate + acceptor
orotate + reduced acceptor
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acceptor: NAD+
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-
?
dihydroorotate + acceptor
orotate + reduced acceptor
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acceptor: menadione
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?
dihydroorotate + acceptor
orotate + reduced acceptor
acceptor: NAD+ for pyrDa gene product, fumarate for pyrDb gene product
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?
dihydroorotate + acceptor
orotate + reduced acceptor
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activity measurement in permeabilized Ehrlich ascites tumor cells, acceptor: nitroblue tetrazolium
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-
?
dihydroorotate + NAD+
orotate + NADH + H+
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fourth step and sole redox reaction in the pyrimidine de novo biosynthetic pathway
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r
dihydroorotate + NAD+
orotate + NADH + H+
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fourth step and sole redox reaction in the pyrimidine de novo biosynthetic pathway
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r
dihydroorotate + NAD+
orotate + NADH + H+
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breakdown of orotate
r
dihydroorotate + NAD+
orotate + NADH + H+
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fourth step and sole redox reaction in the pyrimidine de novo biosynthetic pathway
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r
dihydroorotate + NAD+
orotate + NADH + H+
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fourth step and sole redox reaction in the pyrimidine de novo biosynthetic pathway
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r
dihydroorotate + NAD+
orotate + NADH + H+
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biosynthesis of pyrimidines
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?
dihydroorotate + NAD+
orotate + NADH + H+
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enzyme presumably functions mainly in the metabolic synthesis of pyrimidines
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r
dihydroorotate + NAD+
orotate + NADH + H+
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fourth step and sole redox reaction in the pyrimidine de novo biosynthetic pathway
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r
dihydroorotate + NAD+
orotate + NADH + H+
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fourth step and sole redox reaction in the pyrimidine de novo biosynthetic pathway
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r
additional information
?
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not: uracil, cytosine, 5-methylcytosine, thymine as substrates
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?
additional information
?
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not: uracil, cytosine, 5-methylcytosine, thymine as substrates
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?
additional information
?
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3-acetylpyridine-NAD+ is a more effective oxidant for dihydroorotate than NAD+, methylene blue can serve as oxidant, but not cytochrome c
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?
additional information
?
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NAD+ binding domain in the beta subunit of enzyme, Cys-135 plays a catalytic role and Lys-48 is important for orienting the substrate in the active site and is able to interact with FMN
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?
additional information
?
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O2, fumarate, and NADP+ do not serve as electron acceptors
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?
additional information
?
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O2, fumarate, and NADP+ do not serve as electron acceptors
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-
?
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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
(S)-dihydroorotate + acceptor
orotate + reduced acceptor
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fourth step in UMP-biosynthesis
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?
dihydroorotate + NAD+
orotate + NADH + H+
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fourth step and sole redox reaction in the pyrimidine de novo biosynthetic pathway
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r
dihydroorotate + NAD+
orotate + NADH + H+
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fourth step and sole redox reaction in the pyrimidine de novo biosynthetic pathway
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r
dihydroorotate + NAD+
orotate + NADH + H+
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-
breakdown of orotate
r
dihydroorotate + NAD+
orotate + NADH + H+
-
fourth step and sole redox reaction in the pyrimidine de novo biosynthetic pathway
-
r
dihydroorotate + NAD+
orotate + NADH + H+
-
fourth step and sole redox reaction in the pyrimidine de novo biosynthetic pathway
-
r
dihydroorotate + NAD+
orotate + NADH + H+
-
biosynthesis of pyrimidines
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?
dihydroorotate + NAD+
orotate + NADH + H+
-
enzyme presumably functions mainly in the metabolic synthesis of pyrimidines
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r
dihydroorotate + NAD+
orotate + NADH + H+
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fourth step and sole redox reaction in the pyrimidine de novo biosynthetic pathway
-
r
dihydroorotate + NAD+
orotate + NADH + H+
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fourth step and sole redox reaction in the pyrimidine de novo biosynthetic pathway
-
r
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
4Fe-4S-center
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two [2Fe-2S] clusters as cofactors, tightly bound to the beta subunit and located in the interface of the two dimers centered between the FMN and FAD group, Cys-226, Cys-231, Cys-234 and Cys-249 binds the iron-sulfur cluster
4Fe-4S-center
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cofactor content: 4 non-heme iron and 4 labile sulfur atoms per heterotetramer
4Fe-4S-center
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cofactor content: 4 non-heme iron and 4 labile sulfur atoms per heterotetramer
4Fe-4S-center
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cofactor content: 4 non-heme iron and 4 labile sulfur atoms per heterotetramer
4Fe-4S-center
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cofactor content: 4 non-heme iron and 4 labile sulfur atoms per heterotetramer
FAD
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two tightly bound FAD per heterotetrameric enzyme, FAD is necessary for ability to use NAD+ as electron acceptor, detailed way of binding
flavin
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flavoprotein with two different flavins, probably FAD and FMN
flavin
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interaction of flavin and dihydroorotate is completely dependent on cysteine
FMN
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two tightly bound FMN per heterotetrameric enzyme, detailed way of binding
FMN
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PyrDI is an FMN-containing iron-sulfur flavoprotein, 1 FMN molecule per PyrDI molecule
FMN
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key charge-stabilizing role for Lys-48 of subunit D during reduction of FMN by dihydroorotate or by electron transfer from the 2Fe-2S center
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INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
(S,S)-N-(1-carboxyethyl)-N'-(1-carboxy-2-phenylethyl)urea
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4 mM, 20% inhibition
5-Benzyl-3-(1-carboxy-2-phenylethyl)hydantoin
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time-dependent irreversible inhibition at the active site, mechanism
Hg2+
complete inhibition of both orotate reductase and NADH oxidase reaction
hydantoin
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derived from alpha-amino acids, weak competitive inhibitors, compounds with a benzyl goup are better inhibitors
p-chloromercuribenzoate
complete inhibition of both orotate reductase and NADH oxidase reaction
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
cysteine
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essential for assay, interaction of enzyme with orotate or dihydroorotate is dependent on cysteine, whereas NADH oxidase activity is not
cysteine
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required for activation of purified enzyme, but not if crude extract is used for assay
cysteine
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L-cysteine activates strongly, D-cysteine is also an activator, 1.8 mM L-cysteine is required at pH 8 for half-maximal activity, 10 mM for near-maximal activity
cysteine
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reaction rate increases 2fold in the presence of 2-7 mM cysteine
additional information
-
thiol activation involves reduction of side chain of a cysteine residue corresponding to Cys-130, which functions as a general base
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DISEASE
TITLE OF PUBLICATION
LINK TO PUBMED
Malaria
Purification and characterization of dihydroorotate dehydrogenase from the rodent malaria parasite Plasmodium berghei.
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
22
L-dihydroorotate
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formation of orotate, in absence of NAD+ or NADH, pH 8, 10 mM cysteine
3.97
Orotate
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formation of L-dihydroorotate, in absence of NAD+ or NADH, pH 8, 10 mM cysteine
additional information
additional information
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values not per enzyme molecule, but per mol of flavin
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additional information
additional information
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values not per enzyme molecule, but per mol of flavin
-
additional information
additional information
-
values not per enzyme molecule, but per mol of flavin
-
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
0.06
native wild type enzyme, using 2,6-dichlorophenolindophenol as acceptor, at 30°C
5.9
recombinant wild type enzyme, using 2,6-dichlorophenolindophenol as acceptor, at 37°C
additional information
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