Information on EC 1.3.1.14 - dihydroorotate dehydrogenase (NAD+)

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

EC NUMBER
COMMENTARY hide
1.3.1.14
-
RECOMMENDED NAME
GeneOntology No.
dihydroorotate dehydrogenase (NAD+)
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
(S)-dihydroorotate + NAD+ = orotate + NADH + H+
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dehydrogenation
-
-
-
-
oxidation
-
-
-
-
redox reaction
-
-
-
-
reduction
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Metabolic pathways
-
-
Pyrimidine metabolism
-
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UMP biosynthesis III
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-
pyrimidine metabolism
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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 hide
37255-26-8
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
subunit PyrDb; bifunctional orotate reductase and H2O2-forming NADH oxidase
UniProt
Manually annotated by BRENDA team
Ehrlich ascites tumor cells
-
-
Manually annotated by BRENDA team
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 + 2,6-dichlorophenolindophenol
orotate + reduced 2,6-dichlorophenolindophenol
show the reaction diagram
-
-
-
?
(S)-dihydroorotate + acceptor
orotate + reduced acceptor
show the reaction diagram
(S)-dihydroorotate + NAD+
orotate + NADH + H+
show the reaction diagram
5-fluorodihydroorotate + NAD+
5-fluoroorotate + NADH + H+
show the reaction diagram
-
more active substrate for reverse reaction than orotate
-
-
r
dihydroorotate + acceptor
orotate + reduced acceptor
show the reaction diagram
dihydroorotate + NAD+
orotate + NADH + H+
show the reaction diagram
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
-
fourth step in UMP-biosynthesis
-
?
dihydroorotate + NAD+
orotate + NADH + H+
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4Fe-4S-center
flavin
iron-sulfur centre
-
2FE-2S cluster
additional information
-
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(R,R)-5-benzyl-3-(1-carboxy-2-phenylethyl)hydantoin
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(S)-5-benzyl-3-carboxymethylhydantoin
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(S)-N-carboxymethyl-N'-(1-carboxy-2-phenylethyl)urea
-
-
(S,S)-5-benzyl-3-(1-carboxy-2-phenylethyl)hydantoin
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-
(S,S)-N,N'-bis(1-carboxy-2-phenylethyl)urea
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(S,S)-N-(1-carboxyethyl)-N'-(1-carboxy-2-phenylethyl)urea
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4 mM, 20% inhibition
1,10-phenanthroline
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1 mM, 10% inhibition
2,4-Dihydroxy-6-methyl pyrimidine
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-
3-carboxymethylhydantoin
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-
5-benzyl-3-(1-carboxy-2-phenylethyl)-1-methylhydantoin
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-
5-Benzyl-3-(1-carboxy-2-phenylethyl)hydantoin
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time-dependent irreversible inhibition at the active site, mechanism
5-Methylorotate
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2 mM, 50% inhibition of reduction of orotate
Acriflavin
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partial inhibition
Barbituric acid
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-
cysteine
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inhibitory above 7 mM
Hg2+
complete inhibition of both orotate reductase and NADH oxidase reaction
hydantoin
-
derived from alpha-amino acids, weak competitive inhibitors, compounds with a benzyl goup are better inhibitors
isopropyl hydantoin
-
-
KCN
complete inhibition of orotate reductase reaction
N,N'-bis(carboxymethyl)urea
-
-
Na3PO4
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0.2 M, at pH 6.5, 20% inhibition
NaCl
-
0.2 M, 55% inhibition
p-chloromercuribenzoate
phosphate
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high concentrations
Quinacrine
19.2% inhibition of orotate reductase reaction
additional information
-
not inhibited by 0.01 M arsenite
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-mercaptoethanol
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activates
cysteine
dithiothreitol
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activates
glutathione
Mercaptoacetic acid
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activates
thioglycolate
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stimulating effect, but less effective than cysteine
additional information
-
not activated by L-serine or L-alanine; 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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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.044 - 0.367
(S)-dihydroorotate
0.13
5-Fluoroorotate
-
-
0.09
L-dihydroorotate
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pH 8, 25°C
0.062 - 0.13
NAD+
0.0037
NADH
-
wild-type enzyme
0.0144 - 0.13
Orotate
0.027 - 1
S-dihydroorotate
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.11 - 49.3
(S)-dihydroorotate
22 - 45
L-dihydroorotate
0.021 - 25.7
NADH
0.021 - 25.7
Orotate
additional information
additional information
-
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.9
(R,R)-5-benzyl-3-(1-carboxy-2-phenylethyl)hydantoin
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-
2.3
(S)-5-benzyl-3-carboxymethylhydantoin
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-
0.7
(S)-N-carboxymethyl-N'-(1-carboxy-2-phenylethyl)urea
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-
0.7
(S,S)-5-benzyl-3-(1-carboxy-2-phenylethyl)hydantoin
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-
0.4
(S,S)-N,N'-bis(1-carboxy-2-phenylethyl)urea
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-
4.5
3-carboxymethylhydantoin
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-
0.6
5-benzyl-3-(1-carboxy-2-phenylethyl)-1-methylhydantoin
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-
3.3
isopropyl hydantoin
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-
2.6
N,N'-bis(carboxymethyl)urea
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-
0.0206 - 1
Orotate
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.015
native wild type enzyme, using NAD+ as acceptor, at 30°C
0.05
recombinant wild type enzyme, using NAD+ as acceptor, at 37°C
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
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5
and 6.5, NADH oxidase reaction and dihydrorotate oxidase reaction
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.5 - 7.8
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pH 5.5: 13% of the activity at pH 6.5; pH 7.8: 65% of the activity at pH 6.5
6.4 - 8.9
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below and above the turnover number decreases
6.5
70% of maximal activity, dihydrorotate oxidase reaction
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
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. lactis (strain IL1403)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
28099
-
heterotetramer 2 * 33094 and 2 * 28099 predicted from seqeuence, 2 * 38000 and 2 * 31000, SDS-PAGE
31000
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heterotetramer 2 * 33094 and 2 * 28099 predicted from seqeuence, 2 * 38000 and 2 * 31000, SDS-PAGE
32000
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alpha2,beta2, 2 * 32000 + 2 * 28000, SDS-PAGE
33094
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heterotetramer 2 * 33094 and 2 * 28099 predicted from seqeuence, 2 * 38000 and 2 * 31000, SDS-PAGE
34120
deduced from sequence of cDNA
38000
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heterotetramer 2 * 33094 and 2 * 28099 predicted from seqeuence, 2 * 38000 and 2 * 31000, SDS-PAGE
85000
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native molecular mass of PyrDI
112000 - 124000
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-
114000
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gel filtration; molecular mass of the holoenzyme, calculated mass 122400 Da
120000
-
analytical gel filtration
125000
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gel filtration
130000
gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
heterotetramer
homotetramer
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4 * 30000-31000, guanidine treatment
tetramer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
hanging-drop vapor diffusion
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5
-
more stable than at pH 7.0
390562
7
-
less stable than at pH 6.5
390562
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
dithiothreitol stabilizes
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-18°C, prepared enzyme, that is a yellow powder, several weeks, stable
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-20°C, 0.27 M sodium phosphate, pH 6.3, over 6 months, less than 15% loss of activity
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4°C, crystals, over a month, little or no loss of activity
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4°C, holoenzyme, stable for many days
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above 20°C, 0.2 M phosphate buffer, fast loss of activity
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on ice at room temperature, PyrDI, gradually loses activity over a period of hours
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room temperature, holoenzyme, stable for many hours
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
about 90fold purification
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anion-exchange, gel filtration
-
partial purification
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
; expression in Escherichia coliBL21 DE3, expression of pyrDI alone and coexpression with pyrDII
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expressed in Escherichia coli strain MC1061; two different genes
genes encoding the two alpha and beta polypeptides, expression in Escherichia coli
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C220A
-
pyrDII mutant, conserved, essential for activity
C225A
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pyrDII mutant conserved, essential for activity
C228A
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pyrDII mutant conserved, essential for activity
C230A
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pyrDII mutant nonconserved
C243A
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pyrDII mutant conserved, essential for activity
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
-
KM-value for dihydroorotate is 1.5fold higher than wild-type value, kcat is 448fold lower than wild-type value
K48R
-
KM-value for dihydroorotate is 2fold lower than wild-type value, kcat is 117fold lower than wild-type value
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
activity can be recovered by mixing the purified subunits; PyrDII-containing inclusion bodies are denatured and refolded through dialysis into buffer
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
nutrition
pharmacology
-
drug design based upon selective enzyme inhibition
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