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Information on EC 1.3.5.2 - dihydroorotate dehydrogenase (quinone) and Organism(s) Escherichia coli and UniProt Accession P0A7E1
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1.3.5.2 dihydroorotate dehydrogenase (quinone)IUBMB Comments
This Class 2 dihydroorotate dehydrogenase enzyme contains FMN . The enzyme is found in eukaryotes in the mitochondrial membrane, in cyanobacteria, and in some Gram-negative and Gram-positive bacteria associated with the cytoplasmic membrane [2,5,6]. The reaction is the only redox reaction in the de-novo biosynthesis of pyrimidine nucleotides [2,4]. The best quinone electron acceptors for the enzyme from bovine liver are ubiquinone-6 and ubiquinone-7, although simple quinones, such as benzoquinone, can also act as acceptor at lower rates . Methyl-, ethyl-, tert-butyl and benzyl (S)-dihydroorotates are also substrates, but methyl esters of (S)-1-methyl and (S)-3-methyl and (S)-1,3-dimethyldihydroorotates are not . Class 1 dihydroorotate dehydrogenases use either fumarate (EC 1.3.98.1), NAD+ (EC 1.3.1.14) or NADP+ (EC 1.3.1.15) as electron acceptor.
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Word Map
- 1.3.5.2
- pyrimidine
- falciparum
- plasmodium
- leflunomide
- brequinar
- uridine
- malaria
- arthritis
-
antimalarial
-
rheumatoid
- dihydroorotase
-
salvage
- teriflunomide
-
dhods
- autoimmune
- orotidine
- ump
- atovaquone
- triazolopyrimidine
-
flavoenzyme
- medicine
-
phosphoribosyltransferase
-
antirheumatic
- coq
- drug development
- decylubiquinone
-
dmards
- synthesis
-
species-selective
-
pyre
- pharmacology
-
isoxazole
-
transcarbamoylase
- 5-fluoroorotate
The taxonomic range for the selected organisms is: Escherichia coli
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
Synonyms
dihydroorotate dehydrogenase, pfdhodh, hdhodh, dho-dh, hsdhodh, dihydroorotate dehydrogenase (quinone), 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
DHODH
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
(S)-dihydroorotate + a quinone = orotate + a quinol
enzyme uses a stepwise mechanism for dihydroorotate oxidation
(S)-dihydroorotate + a quinone = orotate + a quinol
existence of 3 families differing in their selectivity for oxidizing substrates: 1A are soluble, containing FMN and use fumarate, 1B are soluble, one subunit contains an iron-sulfur center, FAD and reduces NAD+, family 2 enzymes are membrane-bound, contain FMN and are oxidized by ubiquinone. Mechanism consists of 3 reaction phases. Different binding-mechanisms for enzyme the reaction-steps are suggested
-
(S)-dihydroorotate + a quinone = orotate + a quinol
different binding sites for dihydroorotate and the electron acceptor, two-site ping-pong mechanism. Cleavage site at R182 is conserved between the two major families of dihydroorotate dehydrogenases, it is positioned in a loop, which is crucial for catalysis but irrelevant for protein stability
-
(S)-dihydroorotate + a quinone = orotate + a quinol
the active base S175 and the hydrogen bonding network including residues T178 and F115 work together for efficient deprotonation of dihydroorotate
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
redox reaction
-
-
-
-
oxidation
-
-
-
-
reduction
-
-
-
-
SYSTEMATIC NAME
IUBMB Comments
(S)-dihydroorotate:quinone oxidoreductase
This Class 2 dihydroorotate dehydrogenase enzyme contains FMN [4]. The enzyme is found in eukaryotes in the mitochondrial membrane, in cyanobacteria, and in some Gram-negative and Gram-positive bacteria associated with the cytoplasmic membrane [2,5,6]. The reaction is the only redox reaction in the de-novo biosynthesis of pyrimidine nucleotides [2,4]. The best quinone electron acceptors for the enzyme from bovine liver are ubiquinone-6 and ubiquinone-7, although simple quinones, such as benzoquinone, can also act as acceptor at lower rates [2]. Methyl-, ethyl-, tert-butyl and benzyl (S)-dihydroorotates are also substrates, but methyl esters of (S)-1-methyl and (S)-3-methyl and (S)-1,3-dimethyldihydroorotates are not [2]. Class 1 dihydroorotate dehydrogenases use either fumarate (EC 1.3.98.1), NAD+ (EC 1.3.1.14) or NADP+ (EC 1.3.1.15) as electron acceptor.
CAS REGISTRY NUMBER
COMMENTARY
59088-23-2
-
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-dihydrophenolindophenol
orotate + reduced 2,6-dihydrophenolindophenol
-
-
-
-
r
(S)-dihydroorotate + ubiquinone
orotate + ubiquinol
-
reaction studied with menadione, O2 and ferricyanide
-
?
L-dihydroorotate + 2,6-dichlorophenolindophenol
orotate + reduced 2,6-dichlorophenolindophenol
-
-
-
-
?
(S)-dihydroorotate + acceptor
orotate + reduced acceptor
-
uses ubiquinone as electron acceptor
-
-
?
(S)-dihydroorotate + acceptor
orotate + reduced acceptor
-
with ubiquinone-0, 2,6-dichlorophenolindophenol, menadione, decylubiquinone, fumarate and O2 as electron acceptor
-
?
(S)-dihydroorotate + acceptor
orotate + reduced acceptor
-
fourth step in synthesis of pyrimidine nucleotides
-
?
(S)-dihydroorotate + acceptor
orotate + reduced acceptor
-
fourth step in synthesis of pyrimidine nucleotides
-
?
(S)-dihydroorotate + acceptor
orotate + reduced acceptor
-
fourth step in synthesis of pyrimidine nucleotides
-
?
dihydroorotate + acceptor
orotate + reduced acceptor
-
acceptor: 2,6-dichlorophenolindophenol
-
-
?
dihydroorotate + acceptor
orotate + reduced acceptor
-
acceptor: menaquinone
-
-
?
dihydroorotate + acceptor
orotate + reduced acceptor
-
acceptor: ubiquinone
-
-
?
dihydroorotate + acceptor
orotate + reduced acceptor
-
reaction intermediates, mechanism, pH-dependence of reaction
-
-
?
dihydroorotate + acceptor
orotate + reduced acceptor
-
physiological electron acceptor: ubiquinone (aerobic conditions), menaquinone (anaerobic conditions)
-
-
?
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 + acceptor
orotate + reduced acceptor
-
physiological electron acceptor: ubiquinone (aerobic conditions), menaquinone (anaerobic conditions)
-
-
?
(S)-dihydroorotate + acceptor
orotate + reduced acceptor
-
fourth step in synthesis of pyrimidine nucleotides
-
?
(S)-dihydroorotate + acceptor
orotate + reduced acceptor
-
fourth step in synthesis of pyrimidine nucleotides
-
?
(S)-dihydroorotate + acceptor
orotate + reduced acceptor
-
fourth step in synthesis of pyrimidine nucleotides
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
9.3
(S)-dihydroorotate
-
mutant Y318L, 22°C, pH 7.0, presence of 0.1% TritonX-100
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
electron spin resonance spectra show that the N-terminal binds to membranes and experiences a somewhat high flexibility that could be related to the role of this region as a molecular lid controlling the entrance of the enzyme's active site and thus allowing the enzyme to give access to quinones that are dispersed in the membrane and that are necessary for the catalysis
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
37000
72000
-
sedimentation analysis in sucrose density gradient in presence of detergent
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dimer
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
N111D
large decrease in reduction rate constant. Reduction potential is about 100 mV lower than in wild-type
N172A
large decrease in reduction rate constant. Reduction potential is about 25 mV lower than in wild-type
N172A/N246A
large decrease in reduction rate constant. The maximum flavin absorbance is at 453 nm, blue-shifted 3 nm compared to wild type
N177A
large decrease in reduction rate constant. Reduction potential is about 25 mV lower than in wild-type
F115A
-
mutation slows the rate of flavin reduction by 3 orders of magnitude
F21C/R1C
mutant incorporates into 1-dipalmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/Triton X-100 mixed vesicles and expected to be located right in the core of the more hydrophobic region of the model membrane. Mutated amino acids are either in a strongly immobilized regime or subjected to a fast motion
F5C/R1C
mutant incorporates into 1-dipalmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/Triton X-100 mixed vesicles. Mutated residues experience a high degree of freedom that is compatible with their location in the beginning of the protein chain. Mutated amino acids are either in a strongly immobilized regime or subjected to a fast motion
H19C/R1C
mutant incorporates into 1-dipalmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/Triton X-100 mixed vesicles and expected to be located right in the core of the more hydrophobic region of the model membrane. Mutated amino acids are either in a strongly immobilized regime or subjected to a fast motion
S175A
S175C
-
sufficient activity, catalysis and binding of dihydrooratate are affected
T178A
-
mutation slows the rate of flavin reduction by 3 orders of magnitude. Reduction potential is about 40 mV lower than in wild-type
Y2C/R1C
mutant incorporates into 1-dipalmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/Triton X-100 mixed vesicles. Mutated residues experience a high degree of freedom that is compatible with their location in the beginning of the protein chain
S175A
-
mutation slows the rate of flavin reduction by 3 orders of magnitude. Reduction potential is about 40 mV lower than in wild-type
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20°C, 10 mg/ml, 50 mM sodium phosphate, pH 7.0, 0.1 mM EDTA, 50% glycerol
-
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
overexpression of Escherichia coli dihyroorotate dehyrogenase in same strain, partially deleted for the chromosomal pyrD gene, clone selection followed by ampicillin and by complementation of the pyrimidine requirement
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
-
kinetic isotope effects on flavin reduction in anaerobic stopped-flow experiments, are about 3fold for DHO labeled at the 5-position, about 4fold for DHO labeled at the 6-position, and about 6-7fold for DHO labeled at both the 5- and 6-positions, at a pH value above the pKa controlling reduction, no isotope effect was observed for DHO deuterated at the 5-position, which is consistent with a stepwise reaction, above the kinetic pKa, the deprotonation of C5 is fast enough that it does not contribute to the observed rate constant and, therefore, is not isotopically sensitive
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Rowland, P.; Norager, S.; Jensen, K.F.; Larsen, S.
Crystallization and preliminary x-ray studies of membrane-associated Escherichia coli dihydroorotate dehydrogenase
Acta Crystallogr. Sect. D
56
659-661
2000
Escherichia coli
Bjoernberg, O.; Gruener, A.C.; Roepstorff, P.; Jensen, K.F.
The activity of Escherichia coli dihydroorotate dehydrogenase is dependent on a conserved loop identified by sequence homology, mutagenesis, and limited proteolysis
Biochemistry
38
2899-2908
1999
Escherichia coli, Lactococcus lactis
Palfey, B.A.; Bjoernberg, O.; Jensen, K.F.
Insight into the chemistry of flavin reduction and oxidation in Escherichia coli dihydroorotate dehydrogenase obtained by rapid reaction studies
Biochemistry
40
4381-4390
2001
Escherichia coli
Karibian, D.
Dihydroorotate dehydrogenase (Escherichia coli)
Methods Enzymol.
51
58-63
1978
Escherichia coli
Shi, J.; Palfey, B.A.; Dertouzos, J.; Jensen, K.F.; Gafni, A.; Steel, D.
Multiple states of the Tyr318Leu mutant of dihydroorotate dehydrogenase revealed by single-molecule kinetics
J. Am. Chem. Soc.
126
6914-6922
2004
Escherichia coli
Fagan, R.L.; Nelson, M.N.; Pagano, P.M.; Palfey, B.A.
Mechanism of flavin reduction in class 2 dihydroorotate dehydrogenases
Biochemistry
45
14926-14932
2006
Escherichia coli, Homo sapiens
Fagan, R.L.; Palfey, B.A.
Roles in binding and chemistry for conserved active site residues in the class 2 dihydroorotate dehydrogenase from Escherichia coli
Biochemistry
48
7169-7178
2009
Escherichia coli (P0A7E1), Escherichia coli
Kow, R.L.; Whicher, J.R.; McDonald, C.A.; Palfey, B.A.; Fagan, R.L.
Disruption of the proton relay network in the class 2 dihydroorotate dehydrogenase from Escherichia coli
Biochemistry
48
9801-9809
2009
Escherichia coli
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