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Information on EC 1.3.1.14 - dihydroorotate dehydrogenase (NAD+) and Organism(s) Lactococcus lactis and UniProt Accession A2RJT9
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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
-
1.3.3.1
The taxonomic range for the selected organisms is: Lactococcus lactis
The expected taxonomic range for this enzyme is: Bacteria, Archaea, Eukaryota
The expected taxonomic range for this enzyme is: Bacteria, Archaea, Eukaryota
Synonyms
dhodase, dho dehydrogenase, b-type dihydroorotate dehydrogenase, orotate reductase, dihydro-orotic dehydrogenase, 
more
topSYNONYM 
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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dihydrorotate dehydrogenase B
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-
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L-5,6-dihydro-orotate:NAD oxidoreductase
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orotate reductase
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-
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reductase, orotate
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additional information
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+
detailed thermodynamic analysis, midpoint reduction potential of 2Fe-2S center -212 mV, midpoint reduction potential of FMN -298 mV
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
redox reaction
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oxidation
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reduction
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dehydrogenation
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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.
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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-
-
?
dihydroorotate + acceptor
orotate + reduced acceptor
acceptor: NAD+ for pyrDa gene product, fumarate for pyrDb gene product
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-
?
(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 + 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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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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?
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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?
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
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
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
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
FMN
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two tightly bound FMN per heterotetrameric enzyme, detailed way of binding
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
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
heterotetramer
heterotetramer
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alpha subunit is named PyrDB, beta subunit is named PyrK
heterotetramer
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alpha2,beta2, alpha subunit with 331 amino acid residues, beta subunit with 262 amino acid residues, two closely interacting PyrDB-PyrK dimers
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
K48A
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KM-value for dihydroorotate is 2.2fold higher than wild-type value, kcat is 411fold lower than wild-type value
K48E
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KM-value for dihydroorotate is 4.1fold higher than wild-type value, kcat is 67.5fold lower than wild-type value
K48Q
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KM-value for dihydroorotate is 1.5fold higher than wild-type value, kcat is 448fold lower than wild-type value
K48R
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KM-value for dihydroorotate is 2fold lower than wild-type value, kcat is 117fold lower than wild-type value
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
genes encoding the two alpha and beta polypeptides, expression in Escherichia coli
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Argyrou, A.; Washabaugh, M.W.; Pickart, C.M.
Dihydroorotate dehydrogenase from Clostridium oroticum is a class 1B enzyme and utilizes a concerted mechanism of catalysis
Biochemistry
39
10373-10384
2000
Bacillus subtilis, Enterococcus faecalis, Faecalicatena orotica, Lactococcus lactis
Rowland, P.; Norager, S.; Jensen, K.F.; Larsen, S.
Structure of dihydroorotate dehydrogenase B: electron transfer between two flavin groups bridged by an iron-sulphur cluster
Structure
8
1227-1238
2000
Bacillus subtilis, Enterococcus faecalis, Faecalicatena orotica, Lactococcus lactis
Andersen, P.S.; Jansen, P.J.G.; Hammer, K.
Two different dihydroorotate dehydrogenases in Lactococcus lactis
J. Bacteriol.
176
3975-3982
1994
Lactococcus lactis, Lactococcus lactis (A2RJT9)
Mohsen, A.W.; Rigby, S.E.; Jensen, K.F.; Munro, A.W.; Scrutton, N.S.
Thermodynamic basis of electron transfer in dihydroorotate dehydrogenase B from Lactococcus lactis: analysis by potentiometry, EPR spectroscopy, and ENDOR spectroscopy
Biochemistry
43
6498-6510
2004
Lactococcus lactis
Combe, J.P.; Basran, J.; Hothi, P.; Leys, D.; Rigby, S.E.; Munro, A.W.; Scrutton, N.S.
Lys-D48 is required for charge stabilization, rapid flavin reduction, and internal electron transfer in the catalytic cycle of dihydroorotate dehydrogenase B of Lactococcus lactis
J. Biol. Chem.
281
17977-17988
2006
Lactococcus lactis
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