Information on EC 1.3.1.2 - dihydropyrimidine dehydrogenase (NADP+)

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

EC NUMBER
COMMENTARY hide
1.3.1.2
-
RECOMMENDED NAME
GeneOntology No.
dihydropyrimidine dehydrogenase (NADP+)
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
5,6-dihydrouracil + NADP+ = uracil + NADPH + H+
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
-
-
-
-
redox reaction
-
-
-
-
reduction
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
alanine metabolism
-
-
beta-Alanine metabolism
-
-
Drug metabolism - other enzymes
-
-
Metabolic pathways
-
-
Pantothenate and CoA biosynthesis
-
-
pyrimidine metabolism
-
-
Pyrimidine metabolism
-
-
thymine degradation
-
-
uracil degradation I (reductive)
-
-
SYSTEMATIC NAME
IUBMB Comments
5,6-dihydrouracil:NADP+ 5-oxidoreductase
Also acts on dihydrothymine.
CAS REGISTRY NUMBER
COMMENTARY hide
9029-01-0
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
biotype B, strain ATCC 17536
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
additional information
the N-terminal half of DPD is a member of a family of FAD-containing NADPH oxidoreductases, which transfer electrons to an acceptor protein or domain through [4Fe-4S] clusters of low to very low potential
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
5,6-dihydrouracil + NADP+
uracil + NADPH
show the reaction diagram
5-aminouracil + NADPH
?
show the reaction diagram
5-bromouracil + NADPH
5-bromo-5,6-dihydrouracil + NADP+
show the reaction diagram
5-diazouracil + NADPH
?
show the reaction diagram
-
-
-
-
?
5-fluorouracil + NADPH
5-fluoro-5,6-dihydrouracil + NADP+
show the reaction diagram
5-iodouracil + NADPH
5-iodo-5,6-dihydrouracil + NADP+
show the reaction diagram
5-nitrouracil + NADPH
?
show the reaction diagram
-
-
-
-
?
dihydrofluorouracil + NADP+
5-fluorouracil + NADPH + H+
show the reaction diagram
dihydrothymine + NADP+
thymine + NADPH
show the reaction diagram
dihydrothymine + NADP+
thymine + NADPH + H+
show the reaction diagram
-
-
-
?
dihydrouracil + NADP+
uracil + NADPH + H+
show the reaction diagram
-
-
-
?
hydrouridylic acid + NADPH
?
show the reaction diagram
-
low activity
-
-
?
thymine + NAD(P)H + H+
5,6-dihydrothymine + NAD(P)+
show the reaction diagram
-
-
-
-
?
thymine + NADP+
5,6-dihydrothymine + NADPH + H+
show the reaction diagram
-
-
-
-
?
thymine + NADPH
dihydrothymine + NADP+
show the reaction diagram
uracil + NAD(P)H + H+
5,6-dihydrouracil + NAD(P)+
show the reaction diagram
-
-
-
-
?
uracil + NADP+
5,6-dihydrouracil + NADPH + H+
show the reaction diagram
-
-
-
-
?
uracil + NADPH
5,6-dihydrouracil + NADP+
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
5,6-dihydrouracil + NADP+
uracil + NADPH
show the reaction diagram
5-fluorouracil + NADPH
5-fluoro-5,6-dihydrouracil + NADP+
show the reaction diagram
dihydrofluorouracil + NADP+
5-fluorouracil + NADPH + H+
show the reaction diagram
dihydrothymine + NADP+
thymine + NADPH + H+
show the reaction diagram
Q28943
-
-
-
?
dihydrouracil + NADP+
uracil + NADPH + H+
show the reaction diagram
Q28943
-
-
-
?
thymine + NADPH
dihydrothymine + NADP+
show the reaction diagram
uracil + NADPH
5,6-dihydrouracil + NADP+
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
flavin
NAD+
-
less activity than with NADP+, high concentration necessary
NADH
-
less activity than with NADPH, high concentration necessary
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Fe2+
iron-sulfur cluster binding, overview
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(E)-5-[2-bromovinyl-]uracil
1-deazauracil
-
i.e. 2,6-pyridinediol
2,6-Dihydroxypyridine
-
competitive vs. uracil
3-aminopyridine ADP
-
-
3-deazauracil
-
i.e. 2,4-pyridinediol
5,6-Dihydropyrimidine
-
-
5,6-dihydrouracil
5,6-dioxyuracil
-
i.e. alloxan
5-(phenylselenenyl)uracil
-
-
5-(phenylthio)uracil
-
-
5-benzyloxybenzyluracil
-
-
5-benzyluracil
-
-
5-Bromouracil
5-bromvinyluracil
5-cyanouracil
5-Diazouracil
5-Ethynyluracil
5-Iodo-uracil
5-Iodouracil
5-nitrobarbituric acid
-
-
5-propynyluracil
5-trifluoromethyluracil
alpha-2a-interferon
-
cell lines CAL 27, CAL 33, CAL 51, CAL 7, PANC 3
-
ATP-ribose
Chloramphenicol
-
2.0fold reduced activity in chloramphenicol-treated cells
cisplatin
-
cell lines CAL 27, CAL 33, CAL 51, PANC 3
dipyridamole
-
cell lines CAL 27, CAL 33, CAL 51, ORL 1, PANC 3, mechanism
Hydroxyurea
-
cell lines CAL 27, CAL 33, CAL 51, ORL 1, PANC 3
Uracil
-
-
additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
allopurinol
-
cell lines CAL 27, CAL 51, ORL 1, PANC 3
cisplatin
-
cell line ORL 1
Sulfide
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.24 - 2
5,6-dihydrouracil
0.0011 - 0.0089
5-fluorouracil
0.043 - 0.6
Dihydrothymine
0.193
dihydrouracil
-
-
0.0004 - 0.0038
NADP+
0.00012 - 0.0289
NADPH
0.001 - 0.0068
thymine
0.00012 - 0.0366
Uracil
additional information
additional information
pH dependence and kinetics of recombinant wild-type and mutant enzymes, overview
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.4
5,6-dihydrouracil
Bos taurus
-
-
0.068 - 1.6
Uracil
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
9000 - 42000
NADPH
5000 - 238000
Uracil
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0005
1-deazauracil
-
-
0.0021
3-deazauracil
-
-
0.0066
5,6-dioxyuracil
-
-
0.0048
5-(phenylselenenyl)uracil
-
-
0.0054
5-(phenylthio)uracil
-
-
0.0002
5-benzyloxybenzyluracil
-
-
0.0034
5-benzyluracil
-
-
0.0038
5-nitrobarbituric acid
-
-
0.15
dihydrouracil
-
-
0.001 - 0.011
NADP+
0.007
NADPH
-
-
additional information
additional information
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.00018
-
at 30C
0.0043
-
mutant C671A
0.118
-
cell line CAL 27
0.122
-
cell line CAL 7
0.184
-
cell line CAL 33
0.265
-
cell line PANC 3
0.318
-
cell line CAL 51
0.35
-
-
0.38
-
purified enzyme
0.417 - 0.5
-
purified enzyme
0.43
-
-
2.2
-
substrate hydrothymine
4.7
-
substrate nitouracil
14
-
recombinant from Escherichia coli
20.9
-
substrate 5-iodouracil
21
-
purified enzyme
21.5
-
substrate 5-diazouracil
27.9
-
substrate thymine
34.9
-
substrate uracil
46.9
-
substrate 5-bromouracil
47.4
-
substrate 5-fluorouracil
182.1
-
purified enzyme
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 7.6
-
uracil reduction
7.5
assay at
additional information
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.8 - 8.5
-
-
6.7 - 7.9
-
about 80% of activity maximum at pH 6.7 and 7.9, uracil reduction
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
204000
-
HPLC gel filtration
206000
-
gel filtration
207000 - 210000
-
gel fitration or gradient native PAGE
210000
214000
-
recombinant from E. coli, native PAGE
216000
-
gel filtration
218000
-
gel filtration
220000
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
tetramer
-
4 * 52000, SDS-PAGE
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cocrystallization with 5-iodouracil 1 mM and NADPH 5 mM, ternary complex in 100 mM HEPES, pH 7.5, 22% polyethylene glycol 6000, uracil-4-acetic acid 1 mM, structure determination at different pH-values
-
crystals diffract to 1.9 A
-
mutants E244V and A551T
-
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
45
-
t1/2: 2 min without 2-mercaptoethanol, 2.5 min with 5 mM 2-mercaptoethanol
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
2-mercaptoethanol stabilizes during dialysis and heating
-
24 h dialysis against 35 mM potassium phosphate buffer, pH 7.4, with addition of 5 mM 2-mercaptoethanol or dithiothreitol, stable
-
dithiothreitol stabilizes against inactivation via 5-alkylyluracils
irreversible denaturation of enzyme in dilute solutions below 1 mg/ml
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-15C, 20% loss of activity after 1 month
-
-20C, 2 mg/ml protein, 10% loss of activity after 2 weeks
-
-20C, protein concentration 0.5-1 mg/ml, 1 mM dithiothreitol, stable for several months
-
-80C, 35 mM potassium phosphate, pH 7.4, 1 mM dithioerythritol, more than 2 months without loss of activity, without dithioerythritol 50% loss of activity
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
partial
recombinant protein
-
recombinant wild-type and mutant enzymes from Escherichia coli by affinity chromatography and gel filtration
wild-type and mutant enzyme
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloning of cDNA
-
expression in Escherichia coli
-
expression of wild-type and mutant enzymes in Escherichia coli strain Tuner(DE3)
overproduction of wild-type and mutant enzyme in Escherichia coli
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A551T
natural mutation identified in a patient with complete loss of enzymic activity
E244V
natural mutation identified in a patient with complete loss of enzymic activity
G366A
-
natural mutation, marked decrease in enzyme affinity to NADPH, reduction of Vmax for 5-fluorouracil degrading activity
R235Q
-
the mutation is expected to weaken the binding of the FAD ring system and to change the electronic environment and hence, the redox potential of the cofactor
T768K
-
natural mutation identified in healthy Japanese volunteer, rapid loss of enzyme activity
A551T
-
natural mutation identified in a human with complete loss of enzymic activity. Crystallization data of Sus scrofa recombinant mutant, mutation might prevent binding of the prosthetic group FMN and affect folding of the enzyme protein
C126A
site-directed mutagenesis, a potential [4Fe-4S]-cluster binding residue, the mutant shows slightly increased activity compared to the wild-type enzyme
C671A
-
1% activity compared to that of the wild-type enzyme
E244V
-
natural mutation identified in a human with complete loss of enzymic activity. Crystallization data of Sus scrofa recombinant mutant, mutation interferes with the electron flow between NADPH and the pyrimidine binding site of the enzyme
H673N
site-directed mutagenesis, active site loop residue, the mutant shows reduced activity compared to the wild-type enzyme
H673Q
site-directed mutagenesis, active site loop residue, the mutant shows reduced activity compared to the wild-type enzyme
Q156E
site-directed mutagenesis, [4Fe-4S]-cluster binding residue, inactive mutant
R235A
site-directed mutagenesis, FAD binding residue, inactive mutant
R235K
site-directed mutagenesis, FAD binding residue, inactive mutant
S670A
site-directed mutagenesis, active site loop residue, the mutant shows reduced activity compared to the wild-type enzyme
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
-
study on DPD enzyme expression using RT-PCR, immunohistochemistry, enzymatic activity and ELISA. Highest correlation is observed between protein expression measured by ELISA and enzyme activity, correlation of gene expression and ELISA is also significant
drug development
the enzyme is an adjunct target in cancer therapy since it rapidly breaks down the anti-cancer drug 5-fluorouracil and related compounds
medicine
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