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Information on EC 3.5.2.3 - dihydroorotase
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3.5.2.3 dihydroorotaseSpecify your search results
Word Map
- 3.5.2.3
- pyrimidine
-
transcarbamoylase
- atcase
- phosphoribosyltransferase
- carbamoyl-phosphate
- cpsase
-
carbamoyltransferase
-
glutamine-dependent
- l-dihydroorotate
- orotidine
- dihydro-ouabain
-
succinate-grown
-
pyre
-
n-carbamyl-l-aspartate
- 5-fluoroorotate
- hydantoinase
-
2.1.3.2
- dihydropyrimidinase
- allantoinase
- n-phosphonacetyl-l-aspartate
- drug development
- leflunomide
- synthesis
- medicine
-
amidohydrolases
The enzyme appears in viruses and cellular organisms
Synonyms
BcDHOase, carbamoylaspartic dehydrase, DHO, DHOase, dihydroorotase, dihydroorotate dehydrolase, hDHOase, pyrC, type I DHOase, 
more
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SYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
carbamoylaspartic dehydrase
-
-
-
-
DHO
DHOase
dihydroorotase
dihydroorotate dehydrolase
-
-
-
-
hDHOase
pyrC
type I DHOase
DHOase
-
-
-
-
DHOase
-
the malarial enzyme shares characteristics of both type I and type II DHOases
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
(S)-dihydroorotate + H2O = N-carbamoyl-L-aspartate
-
-
-
-
(S)-dihydroorotate + H2O = N-carbamoyl-L-aspartate
unique catalytic mechanism
-
(S)-dihydroorotate + H2O = N-carbamoyl-L-aspartate
complex formation of enzyme with aspartate transcarbamoylase drives reaction in the biosynthetic direction
-
(S)-dihydroorotate + H2O = N-carbamoyl-L-aspartate
catalytic mechanism, overview
-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
formation of cyclic amides
-
-
-
-
hydrolysis of cyclic amides
-
-
-
-
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PATHWAY SOURCE
PATHWAYS
BRENDA
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SYSTEMATIC NAME
IUBMB Comments
(S)-dihydroorotate amidohydrolase
-
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CAS REGISTRY NUMBER
COMMENTARY
9024-93-5
-
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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
dexrazoxane + H2O
ADR-925
-
i.e. ICRF187 or (+)-1,2-bis(3,5-dioxopiperazin-1-yl)propane, cardioprotective agent
i.e. N,N’-[(1S)-1-methyl-1,2-ethanediyl]bis[N-(2-amino-2-oxoethyl)glycine], biologically active form
?
N-(2-amino-2-oxoethyl)-N-[(1S)-2-(3,5-dioxo-1-piperazinyl)-1-methylethyl]glycine + H2O
N,N'-[(1S)-1-methyl-1,2-ethanediyl]bis[N-(2-amino-2-oxoethyl)]glycine
-
-
-
-
ir
N-(2-amino-2-oxoethyl)-N-[(2S)-2-(3,5-dioxo-1-piperazinyl)propyl]glycine + H2O
N,N'-[(1S)-1-methyl-1,2-ethanediyl]bis[N-(2-amino-2-oxoethyl)]glycine
-
-
-
-
ir
(S)-dihydroorotate + H2O
N-carbamoyl-L-aspartate
-
aspartate transcarbamoylase-dihydroorotase complex
-
-
r
(S)-dihydroorotate + H2O
N-carbamoyl-L-aspartate
-
aspartate transcarbamoylase-dihydroorotase complex
-
-
r
(S)-dihydroorotate + H2O
N-carbamoyl-L-aspartate
-
absolute substrate specificity
-
-
?
L-carbamoyl-L-aspartate
L-dihydroorotate + H2O
-
-
-
-
r
L-carbamoylaspartate
L-dihydroorotate + H2O
-
-
-
-
r
additional information
?
-
no substrate: hydantoin, dihydrouracil, phthalimide, and 3-imonoisoindolinone
-
-
-
additional information
?
-
-
no substrate: hydantoin, dihydrouracil, phthalimide, and 3-imonoisoindolinone
-
-
-
additional information
?
-
-
allantoin, hydantoin, and phthalimide are not hydrolyzed by dihydroorotase
-
-
-
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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
L-carbamoyl-L-aspartate
L-dihydroorotate + H2O
-
-
-
-
r
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Zn2+
Zn2+
-
tagged enzyme contains ca. 1.0 zinc atom per subunit measured by atomic absorption spectroscopy
Zn2+
dihydroorotase enzyme is a di-metalloenzyme that is dependent upon two zinc atoms in the active site for activity
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INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
(S)-1,2,3,6-tetrahydro-6-oxopyridine-2-carboxylic acid
-
competitive inhibitor versus dihydroorotate and thio-dihydroorotate
4(R)-hydroxy-6-oxo-piperidine-2(S)-carboxylic acid
-
competitive inhibitor versus dihydroorotate and thio-dihydroorotate
4(S)-hydroxy-6-oxo-piperidine-2(S)-carboxylic acid
-
competitive inhibitor versus dihydroorotate and thio-dihydroorotate
4,6-dioxo-piperidine-2-(S)-carboxylic acid
-
most active competitive inhibitor versus dihydroorotate and thio-dihydroorotate
desferrioxamine
-
hypoxia causes a decrease in carbamoyl phosphate synthetase-aspartate carbamoyltransferase-dihydroorotase expression incubated under desferrioxamine-induced HIF-1alpha accumulation detected in A293T, IMR32, colo320DM and HeLa cell lines
diethyl dicarbonate
-
strong inhibition at 1 mM, activity is restored more than 95% when the enzyme is preincubated with both 1 mM diethyl dicarbonate and 5 mM L-carbamoyl-L-aspartate
2-oxo-1,2,3,6-tetrahydropyrimidine-4,6-dicarboxylic acid
-
at 37°C or 60°c the enzyme binds 2-oxo-1,2,3,6-tetrahydropyrimidine-4,6-dicarboxylic acid only slightly more strongly than DHO; competitive inhibitor
2-oxo-1,2,3,6-tetrahydropyrimidine-4,6-dicarboxylic acid
-
at 37°C or 60°C the enzyme binds 2-oxo-1,2,3,6-tetrahydropyrimidine-4,6-dicarboxylic acid more tightly than DHO
2-oxo-1,2,3,6-tetrahydropyrimidine-4,6-dicarboxylic acid
-
competitive inhibitor
additional information
-
hypoxia causes a decrease in carbamoyl phosphate synthetase-aspartate carbamoyltransferase-dihydroorotase expression detected in A293T, IMR32, HeLa cell lines and human endometiral stromal cells
-
additional information
-
binding and inhibition of allantoinase dihydroorotase by flavonols and the substrates of other cyclic amidohydrolases, dissociation constants, docking analysis using three-dimensional structure model, PDB ID 3JZE, overview. Hydantoin and allantoin bind to dihydroorotase, but do not affect its activity, no inhibition by phthalimide
-
additional information
-
EDTA and 1,10-phenanthroline have no effect on the enzyme activity; L-carbamoyl-L-aspartate and L-dihydroorotate have no inhibitory effect on the enzyme at the pH 7.4
-
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
-
following pyrimidine limitation of orotidine 5'-monophosphate decarboxylase mutant strain cells, dihydroorotase and dihydroorotate dehydrogenase activities double while aspartate transcarbamoylase and orotate phosphoribosyltransferase activities are slightly elevated compared to their activities in the mutant strain cells grown on excess uracil
-
Dimethylsulfoxide
-
increases the catalytic efficiency on the ring cyclization reaction, but not on the ring hydrolysis
Dimethylsulfoxide
-
increases the catalytic efficiency on the ring cyclization reaction, but not on the ring hydrolysis
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DISEASE
TITLE OF PUBLICATION
LINK TO PUBMED
Anemia, Hypoplastic, Congenital
Elevation of pyrimidine enzyme activities in the RBC of patients with congenital hypoplastic anaemia and their parents.
Arthrogryposis
Distal Humerus External Rotation Osteotomy for Hand Position in Arthrogryposis.
Carcinoma
Interconversion of carbamayl-L-aspartate and L-dihydroorotate by dihydroorotase from mouse Ehrlich ascites carcinoma.
Carcinoma
The effects of pH and inhibitors upon the catalytic activity of the dihydroorotase of multienzymatic protein pyr1-3 from mouse Ehrlich ascites carcinoma.
Carcinoma, Hepatocellular
Phosphorylation and dephosphorylation of carbamoyl-phosphate synthetase II complex of rat ascites hepatoma cells.
Carcinoma, Hepatocellular
Purification of homogeneous glutamine-dependent carbamyl phosphate synthetase from ascites hepatoma cells as a complex with aspartate transcarbamylase and dihydroorotase.
Carcinoma, Hepatocellular
STUDIES ON DIHYDROOROTASE ACTIVITY IN PREPARATIONS FROM NOVIKOFF ASCITES HEPATOMA CELLS.
Chagas Disease
Molecular interaction of the first 3 enzymes of the de novo pyrimidine biosynthetic pathway of Trypanosoma cruzi.
dihydroorotate dehydrogenase (fumarate) deficiency
Elevated plasma dihydroorotate in Miller syndrome: Biochemical, diagnostic and clinical implications, and treatment with uridine.
hypoxanthine phosphoribosyltransferase deficiency
Elevated aspartate transcarbamylase and dihydroorotase activities in erythrocytes from patients with hypoxanthine guanine phosphoribosyltransferase deficiency.
Infections
Vector capacity of Rhipicephalus appendiculatus and Amblyomma variegatum for Thogoto and Dhori viruses.
Influenza, Human
Sequence analyses of Thogoto viral RNA segment 3: evidence for a distant relationship between an arbovirus and members of the Orthomyxoviridae.
Leptospirosis
Control and prevention of rat fever (Leptospirosis) outbreak in six villages of Raichur district, Karnataka.
Lesch-Nyhan Syndrome
Elevated aspartate transcarbamylase and dihydroorotase activities in erythrocytes from patients with hypoxanthine guanine phosphoribosyltransferase deficiency.
Malaria
Cytotoxic effects of inhibitors of de novo pyrimidine biosynthesis upon Plasmodium falciparum.
Malaria
The interaction of 5-fluoroorotic acid with transition metals: synthesis and characterisation of Ni(II), Cu(II) and Zn(II) complexes.
Measles
The quality of immunization data from routine primary health care reports: a case from Nepal.
Neoplasms
Diversion of aspartate in ASS1-deficient tumours fosters de novo pyrimidine synthesis.
Neoplasms
Targeting pyrimidine synthesis accentuates molecular therapy response in glioblastoma stem cells.
Poliomyelitis
The quality of immunization data from routine primary health care reports: a case from Nepal.
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.118
(S)-dihydroorotate
pH 8.3, 25°C, recombinant detagged enzyme
0.167
(S)-dihydroorotate
pH 8.3, 25°C, recombinant tagged enzyme
6
L-carbamoyl-L-aspartate
-
mutant enzyme T109S, at pH 5.8; mutant enzyme T110V, at pH 5.8
0.334
N-Carbamoyl-L-aspartate
pH 8.3, 25°C, recombinant tagged enzyme
0.348
N-Carbamoyl-L-aspartate
pH 8.3, 25°C, recombinant detagged enzyme
additional information
carbamoyl aspartate
-
mutants D250A, D250H, D250N, R20Q, R20M, N44A, H254N inactive
additional information
additional information
-
Michaelis-Menten kinetics
-
additional information
additional information
forward and reverse kinetic rate constants for both tagged and wild-type enzyme, kinetic analysis, overview
-
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
1.55
(S)-dihydroorotate
pH 8.3, 25°C, recombinant detagged enzyme
1.9
N-Carbamoyl-L-aspartate
pH 8.3, 25°C, recombinant tagged enzyme
2.48
N-Carbamoyl-L-aspartate
pH 8.3, 25°C, recombinant detagged enzyme
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
13.1
(S)-dihydroorotate
pH 8.3, 25°C, recombinant detagged enzyme
5.69
N-Carbamoyl-L-aspartate
pH 8.3, 25°C, recombinant tagged enzyme
7.13
N-Carbamoyl-L-aspartate
pH 8.3, 25°C, recombinant detagged enzyme
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
2.3
(S)-1,2,3,6-tetrahydro-6-oxopyridine-2-carboxylic acid
-
with thio-dihydroorotate as substrate, pH. 8.0
1.6
4(R)-hydroxy-6-oxo-piperidine-2(S)-carboxylic acid
-
with thio-dihydroorotate as substrate, pH. 8.0
3
4(S)-hydroxy-6-oxo-piperidine-2(S)-carboxylic acid
-
with thio-dihydroorotate as substrate, pH. 8.0
3.5
5-iodoorotate
-
Ki above 3.5 mM, for the ring cleavage reaction; Ki above 3.5 mM, for the ring cyclization reaction
0.0045
2-oxo-1,2,3,6-tetrahydropyrimidine-4,6-dicarboxylic acid
-
at 37°C
0.083
2-oxo-1,2,3,6-tetrahydropyrimidine-4,6-dicarboxylic acid
-
at 37°C
0.102
2-oxo-1,2,3,6-tetrahydropyrimidine-4,6-dicarboxylic acid
-
at 60°C
0.076
4,6-dioxo-piperidine-2-(S)-carboxylic acid
-
with dihydroorotate as substrate, pH. 7.0
0.096
4,6-dioxo-piperidine-2-(S)-carboxylic acid
-
with thio-dihydroorotate as substrate, pH. 7.0
0.12
4,6-dioxo-piperidine-2-(S)-carboxylic acid
-
with thio-dihydroorotate as substrate, pH. 8.0
0.21
4,6-dioxo-piperidine-2-(S)-carboxylic acid
-
with dihydroorotate as substrate, pH. 8.0
0.32
4,6-dioxo-piperidine-2-(S)-carboxylic acid
-
with thio-dihydroorotate as substrate, pH. 9.0
0.44
4,6-dioxo-piperidine-2-(S)-carboxylic acid
-
with dihydroorotate as substrate, pH. 9.0
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IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
25
37
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TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
25 - 45
37 - 60
25 - 45
-
; differences in kinetic isotope effects due to temperature change are nonexistent for hDHOase even though both systems are run at suboptimal temperatures
37 - 60
-
; kinetic isotope effects due to temperature change show a very moderate difference in the secondary kinetic isotope effect for BcDHOase even though both systems are run at suboptimal temperatures
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pI VALUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain RH lambda Zap II cDNA library, strain PC117 and 5TN; strains RH, PC117, and 5TN
SwissProt
brenda
in noncovalent association with aspartate transcarbamoylase, possible model for the mammalian polypeptide chain CPSase/ATCase/DHOase during pyrimidine biosynthesis
UniProt
brenda
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SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
; maintained by serial passage in the peritoneal cavity of CFW mice
brenda
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LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
metabolism
physiological function
-
following pyrimidine limitation of orotidine 5'-monophosphate decarboxylase mutant strain cells, dihydroorotase and dihydroorotate dehydrogenase activities double while aspartate transcarbamoylase and orotate phosphoribosyltransferase activities are slightly elevated compared to their activities in the mutant strain cells grown on excess uracil
additional information
-
the enzyme contains four histidine, one aspartate, and one post-carboxylated lysine residue, which are required for metal binding and catalytic activity
metabolism
third enzyme in the bacterial de novo pyrimidine biosynthesis pathway, overview
metabolism
-
third enzyme in the bacterial de novo pyrimidine biosynthesis pathway, overview
-
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PDB
SCOP
CATH
UNIPROT
ORGANISM
Aquifex aeolicus (strain VF5)
Burkholderia cenocepacia (strain ATCC BAA-245 / DSM 16553 / LMG 16656 / NCTC 13227 / J2315 / CF5610)
Q0PBP6
Campylobacter jejuni subsp. jejuni serotype O:2 (strain ATCC 700819 / NCTC 11168)
Escherichia coli (strain ATCC 8739 / DSM 1576 / Crooks)
Escherichia coli (strain K12)
Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Vibrio cholerae serotype O1 (strain ATCC 39315 / El Tor Inaba N16961)
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
38000
40000
42000
45600
gel filtration and laser light scattering; gel filtration, laser light scattering
48090
-
electrospray ionization mass spectroscopy, enzyme with hexahistidine tag; mass spectroscopy, BcDHOase with the hexahistidine tag
49000
498000
-
gel filtration, DHOase in complex with aspartate transcarbamoylase
500000
-
aspartate transcarbamoylase-dihydroorotase complex, gel filtration
49000
-
dihydroorotase, electro-elution, SDS-PAGE and Coomassie staining
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SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dimer
hexamer
homodimer
homotetramer
-
temperature-dependent conversion to homodimer, crystallization data
monomer
multimer
additional information
homodimer
-
crystallization data, asymmetry between active sites, with N-carbamyl-L-aspartate bound to one site and dihydroorotate bound to the other
homodimer
-
temperature-dependent conversion to homotetramer, crystallization data
monomer
-
1 * 40000, gel filtration, 75% of the total activity is associated with the monomeric form
monomer
1 * 44200, SDS-PAGE; 1 * 45600, gel filtration and laser light scattering
multimer
-
multi-enzyme complex, carbamoyl phosphate synthetase, aspartate transcarbamylase and dihydroorotase, 129000 + 39000 + 44000, SDS-PAGE
additional information
-
recombinant protein lacks catalytic activity, activity is acquired by forming a complex with aspartate transcarbamoylase, complex may be a heterohexamer or dodecamer
additional information
the His-SUMO tag does not interfere with SaPyrC dimerization
additional information
-
the His-SUMO tag does not interfere with SaPyrC dimerization
-
additional information
-
direct intermolecular interactions between carbamoylphosphate synthetase II, aspartate transcarbamoylase, and dihydroorotase, which catalyze the first 3 reaction steps of the de novo pyrimidine biosynthetic pathway
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CRYSTALLIZATION/commentary
ORGANISM
UNIPROT
LITERATURE
hanging drop method, monoclinic structure has a novel cysteine ligand to the zinc that blocks the active site and functions as a cysteine switch, active site residues are located in disordered loops, which may function as a disorder-to-order entropy switch
to 2.6 A resolution. The overall tertiary structure formed from the TIM-barrel secondary structure motif is conical, with the active site residing at the base of the cone. The active site includes a binuclear Zn center, with two histidine (His59 and His61) and two asparagine (Asp151 and Asp304) residues bound to the more buried first Zn atom and two histidine (178 and231) residues and Asp151 bound to the second Zn atom. The carboxylate side group of Asp151 forms a bridge between the two Zn atoms
crystallized without ligand and in the presence of the inhibitors 2-oxo-1,2,3,6-tetrahydropyrimidine-4,6-dicarboxylate and 5-fluoroorotate, hanging drop vapour diffusion method with 15-20% (w/v) polyethylene glycol 3350, 0.1 M MES (pH 6.0-6.5), 75 mM MgCl2, 150 mM KCl (with ligand) or 20-25% polyethylene glycol 3350, 0.1 M Na HEPES (pH 7) and 0.2 M NaF (without ligand)
-
hanging drop vapour diffusion method with 15-20% polyethylene glycol 3350, 0.1 M MES, pH 6.0-6.5, 75 mM MgCl2, and 150 mM KCl
-
in complex with 5-fluoroorotate, hanging drop vapour diffusion method 14-16% PEG 3350, 0.1 M MES pH 6.25, 25 mM MgCl2, 0.2 M KCl and 30% sucrose
-
in the presence of enantiomerically pure L-dihydroorotate, refined at 1.9 Angstrom resolution
-
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
C221S/C263S/C265S/C268S
-
this quadruple mutant is relatively stable to oxidation and has kinetic parameters consistent with reported values for wild type DHO
T109S
additional information
additional information
in noncovalent association with aspartate transcarbamoylase, possible model for mammalian polypeptide chain CPSase/ATCase/DHOase during pyrimidine biosynthesis
additional information
-
deletion mutant DELTA107-116 DHOase shows negligible activity
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
additional information
stability decreases in the order pH 8 pH pH 6 pH 9
713469
additional information
-
stability decreases in the order pH 8 pH pH 6 pH 9
713469
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
50
70
99
-
enzyme complex with aspartate transcarbamoylase, both activities are stable
70
-
BcDHOase starts to unfold at temperatures greater than 70°C with a melting temperature of 79.5°C
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4°C, under nitrogen, 100 mM Tris phosphate buffer, pH 7, metal free 10 mM N-carbamoyl-DL-aspartate, at least 2 months
-
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PURIFICATION/commentary
ORGANISM
UNIPROT
LITERATURE
by Ni-NTA chromatography and Poros Q anion-exchange column; Ni-NTA agarose column chromatography, Poros Q column chromatography, and Sephadex G-75 gel filtration
-
DEAE-Sephacel column chromatography; recombinant protein purified to homogeneity
dihydroorotase and aspartate transcarbamoylase copurify, partially purified by anion exchange chromatography, gel filtration, hydrophobic interaction chromatography; HiTrap Q column chromatography, Hiload Superdex gel filtration, and phenyl-Superose HR column chromatography
-
N-linked butylamine-agarose column chromatography and DEAE-Sephacel column chromatography
-
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and dialysis
-
recombinant N-terminal His-SUMO-tagged enzyme SaPyrC from Escherichia coli strain BL21(DE3), by nickel affinity chromatography, dialysis, and His-SUMO tag cleavage through HRV 3C protease, another step of nickel affinity chromatography to remove the tag, followed by ultrafiltration, and gel filtration, to about 95% purity, method optimization, the His-SUMO tag affects enzyme activity slightly
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CLONED/commentary
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21 cells; overexpression in Escherichia coli BL21(DES/pLysS)
-
expressed in Escherichia coli strain SO1263; overexpression in Escherichia coli strain BL21(DE3)
-
expressed in Escherichia coli strain SO1263; overexpression in Escherichia coli strain SØ1263/pyrC-
-
expressed in Escherichia coli strain X7014; expression in Escherichia coli mutant strain SØ1263 and strain X7014
expression in Escherichia coli
expression in Escherichia colipyrC mutant strain X7014a, genes pyrC(PA3527) and pyrC2(PA5541) can complement DHOase mutant, pyrC constitutively expressed, pyrC2 can only complement the DHOase mutant when expressed from the lacZalpha promotor
expression of yeast-hamster chimeric proteins in Escherichia coli
gene pyrC, DNA and amino acid sequence determination and analysis, sequence comparison, expression of N-terminal His-SUMO-tagged enzyme in Escherichia coli strain BL21(DE3) with an HRV 3C protease recognition site inserted between the SUMO tag and SaPyrC to allow for improved cleavage by HRV protease, method optimization
recombinant overexpression of His-tagged enzyme in Escherichia coli strain BL21(DE3)
-
with a fusion peptide containing six histidine residues at the amino terminus
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
drug development
medicine
synthesis
-
growth conditions which maintain DHOase overproduction require supplementation of Yeast Carbon Base with asparagines 5 g/l, glucose 20 g/l at pH 5.5. In this medium DHOase activity decreases with time, thus in terms of DHOase yield, the best time for harvesting cells is reached after 30 to 38 h of growth
drug development
the essential enzyme is a target for antibacterial drug design
drug development
-
the essential enzyme is a target for antibacterial drug design
-
medicine
-
activation of the cardioprotective agent dexrazoxane by the enzyme in heart, mechanism
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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Coleman, P.F.; Suttle, D.P.; Stark, G.R.
Purification of a multifunctional protein bearing carbamyl-phosphate synthase, aspartate transcarbamylase, and dihydroorotase enzyme activities from mutant hamster cells
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51
121-134
1978
Cricetinae
brenda
Mori, M.; Tatibana, M.
A multienzyme complex of carbamoyl-phosphate synthase (glutamine):aspartate carbamoyltransferase:dihydroorotase (rat ascites hepatoma cells and rat liver)
Methods Enzymol.
51
111-121
1978
Rattus norvegicus
brenda
Christopherson, R.I.; Williams, N.K.; Schoettle, S.L.; Szabados, E.; Hambley, T.W.; Manthey, M.K.
Inhibitors of dihydroorotase, amidophosphoribosyltransferase and IMP cyclohydrolase as potential drugs
Biochem. Soc. Trans.
23
888-893
1995
Cricetinae
brenda
Mukherjee, T.; Ray, M.; Bhaduri, A.
Aspartate transcarbamylase from Leishmania donovani
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263
708-713
1988
Leishmania donovani
brenda
Carrey, E.A.; Hardie, D.G.
Mapping of catalytic domains and phosphorylation sites in the multifunctional pyrimidine-biosynthetic protein CAD
Eur. J. Biochem.
171
583-588
1988
Cricetinae
brenda
Kelly, R.E.; Mally, M.I.; Evans, D.R.
The dihydroorotase domain of the multifunctional protein CAD
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261
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1986
Macaca mulatta polyomavirus 1
brenda
Washabough, M.W.; Collins, K.D.
Dihydroorotase from Escherichia colii. Sulfhydryl group-metal ion interactions
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261
5920-5929
1986
Escherichia coli
brenda
Pettigrew, D.W.; Mehta, B.J.; Bidigare, R.R.; Choudhuri, R.R.; Scheffler, J.F.; Sander, E.G.
Enzyme elements involved in the interconversion of L-carbamylaspartate and L-dihydroorotate by dihydroorotase from Clostridium oroticum
Arch. Biochem. Biophys.
243
447-553
1985
Faecalicatena orotica
brenda
Bidigare, R.R.; Sander, E.G.; Pettigrew, D.W.
Evidence for a pH-dependent isomerization of Clostridium oroticum dihydroorotase
Biochim. Biophys. Acta
831
159-160
1985
Faecalicatena orotica
brenda
Pettigrew, D.W.; Bidigare, R.R.; Mehta, B.J.; Williams, M.I.; Sander, E.G.
Dihydro-orotase from Clostridium oroticum
Biochem. J.
230
101-108
1985
Faecalicatena orotica
brenda
Washabaugh, M.W.; Collins, K.D.
Dihydroorotase from Escherichia coli. Purification and characterization
J. Biol. Chem.
259
3293-3298
1984
Escherichia coli
brenda
Davidson, J.N.; Rumsby, P.C.; Tamaren, J.
Organization of a multifunctional protein in pyrimidine biosynthesis
J. Biol. Chem.
256
5220-5225
1981
Cricetinae
brenda
Hammond, D.J.; Gutteridge W.E.
Enzymes of pyrimidine biosynthesis in Trypanosoma cruzi
FEBS Lett.
118
259-262
1980
Trypanosoma cruzi
brenda
Scheffler, J.E.; MA, J.; Sander E.G.
Dihydroorotase from Clostridium oroticum is an allosteric enzyme
Biochem. Biophys. Res. Commun.
91
563-568
1979
Faecalicatena orotica
brenda
Christopherson, R.I.; Jones, M.E.
Interconvension of carbamyl-L-aspartate and L-dihydroorotate by dihydroorotase from mouse Ehrlich ascites carcinoma
J. Biol. Chem.
254
12506-12512
1979
Mus musculus
brenda
Taylor, W.H.; Taylor, M.L.; Balch, W.E.; Gilchrist, P.S.
Purification and properties of dihydroorotase, a zinc-containing metalloenzyme in Clostridium oroticum
J. Bacteriol.
127
863-873
1976
Faecalicatena orotica
brenda
Kennedy, J.
Dihydroorotase from rat liver: purification, properties and regulatory role in pyrimidine biosynthesis
Arch. Biochem. Biophys.
160
358-365
1974
Rattus norvegicus
brenda
Serre, V.; Guy, H.; Liu, X.; Penverne, B.; Herve, G.; Evans, D.
Allosteric regulation and substrate channeling in multifunctional pyrimidine biosynthetic complexes: analysis of isolated domains and yeast-mammalian chimeric proteins
J. Mol. Biol.
281
363-377
1998
Cricetinae, Saccharomyces cerevisiae
brenda
Krungkrai, J.; Cerami, A.; Henderson, G.B.
Pyrimidine biosynthesis in parasitic protozoa: purification of a monofunctional dihydroorotase from Plasmodium berghei and Crithidia fasciculata
Biochemistry
29
6270-6275
1990
Crithidia fasciculata, Plasmodium berghei
brenda
Krungkrai, J.; Krungkrai, S.R.; Phakanont, K.
Antimalarial activity of orotate analogs that inhibit dihydroorotase and dihydroorotate dehydrogenase
Biochem. Pharmacol.
43
1295-1301
1992
Plasmodium berghei
brenda
Williams, N.K.; Manthey, M.K.; Hambley, T.W.; O'Donoghue, S.I.; Keegan, M.; Chapman, B.E.; Christopherson, R.I.
Catalysis by hamster dihydroorotase: zinc binding, site-directed mutagenesis, and interaction with inhibitors
Biochemistry
34
11344-11352
1995
Cricetinae
brenda
Ogawa, J.; Shimizu, S.
Purification and characterization of dihydroorotase from Pseudomonas putida
Arch. Microbiol.
164
353-357
1995
Pseudomonas putida
brenda
Williams, N.K.; Peide, Y.; Seymour, K.K.; Ralston, G.B.; Christopherson, R.I.
Expression of catallytically active hamster dihydroorotase domain in Escherichia coli: purification and characterization
Protein Eng.
6
333-340
1993
Cricetinae
brenda
Aleksenko.A.; Liu, W.; Gojkovic, Z.; Nielsen, J.; Piskur, J.
Structural and transcriptional analysis of the pyrABCN, pyrD and pyrF genes in Aspergillus nidulans and the evolutionary origin of fungal dihydroorotases
Mol. Microbiol.
33
599-611
1999
Aspergillus nidulans
brenda
Purcarea, C.; Martin, P.; Vickrey, J.F.; Guy, H.I.; Edwards, B.F.; Evans, D.R.
Cloning, expression and preliminary X-ray analysis of the dihydroorotase from the hyperthermophilic eubacterium Aquifex aeolicus
Acta Crystallogr. Sect. D
58
154-156
2002
Aquifex aeolicus
brenda
Maher, M.J.; Huang, D.T.; Guss, J.M.; Collyer, C.A.; Christopherson, R.I.
Crystallization of hamster dihydroorotase: involvement of a disulfide-linked tetrameric form
Acta Crystallogr. Sect. D
59
381-384
2003
Mesocricetus auratus
brenda
Thoden, J.B.; Phillips, G.N., Jr.; Neal, T.M.; Raushel, F.M.; Holden, H.M.
Molecular structure of dihydroorotase: a paradigm for catalysis through the use of a binuclear metal center
Biochemistry
40
6989-6997
2001
Escherichia coli
brenda
Schroeder, P.E.; Davidson, J.N.; Hasinoff, B.B.
Dihydroorotase catalyzes the ring opening of the hydrolysis intermediates of the cardioprotective drug dexrazoxane (ICRF-187)
Drug Metab. Dispos.
30
1431-1435
2002
Mesocricetus auratus
brenda
Ahuja, A.; Purcarea, C.; Ebert, R.; Sadecki, S.; Guy, H.I.; Evans, D.R.
Aquifex aeolicus dihydroorotase: association with aspartate transcarbamoylase switches on catalytic activity
J. Biol. Chem.
279
53136-53144
2004
Aquifex aeolicus
brenda
Brichta, D.M.; Azad, K.N.; Ralli, P.; O'Donovan, G.A.
Pseudomonas aeruginosa dihydroorotases: a tale of three pyrCs
Arch. Microbiol.
182
7-17
2004
Pseudomonas aeruginosa (P72170), Pseudomonas aeruginosa
brenda
McPhail, D.; Shepherdson, M.
The aspartate transcarbamoylase-dihydroorotase complex in Deinococcus radiophilus has an active dihydroorotase
Arch. Microbiol.
185
78-81
2006
Deinococcus radiophilus, Deinococcus radiophilus NCIMB 10648
brenda
Porter, T.N.; Li, Y.; Raushel, F.M.
Mechanism of the dihydroorotase reaction
Biochemistry
43
16285-16292
2004
Escherichia coli (P05020)
brenda
Anderson, M.A.; Cleland, W.W.; Huang, D.T.; Chan, C.; Shojaei, M.; Christopherson, R.I.
13C and 15N isotope effects for conversion of L-dihydroorotate to N-carbamyl-L-aspartate using dihydroorotase from hamster and Bacillus caldolyticus
Biochemistry
45
7132-7139
2006
Bacillus caldolyticus, Mesocricetus auratus
brenda
Huang, D.T.; Kaplan, J.; Menz, R.I.; Katis, V.L.; Wake, R.G.; Zhao, F.; Wolfenden, R.; Christopherson, R.I.
Thermodynamic analysis of catalysis by the dihydroorotases from hamster and Bacillus caldolyticus, as compared with the uncatalyzed reaction
Biochemistry
45
8275-8283
2006
Bacillus caldolyticus, Cricetinae, Mesocricetus auratus
brenda
Li, Y.; Raushel, F.M.
Inhibitors designed for the active site of dihydroorotase
Bioorg. Chem.
33
470-483
2005
Escherichia coli
brenda
Lee, M.; Chan, C.W.; Mitchell Guss, J.; Christopherson, R.I.; Maher, M.J.
Dihydroorotase from Escherichia coli: loop movement and cooperativity between subunits
J. Mol. Biol.
348
523-533
2005
Arabidopsis thaliana (O04904), Escherichia coli, Escherichia coli (P05020), Helicobacter pylori (P56465), Homo sapiens (P27708), Mesocricetus auratus (P08955), Plasmodium falciparum (Q8IKA9), Saccharomyces cerevisiae (P20051)
brenda
Martin P.D; Purcarea, C.; Zhang, P.; Vaishnav, A.; Sadecki, S.; Guy-Evans, H.I.; Evans, D.R.; Edwards, B.F.
The crystal structure of a novel, latent dihydroorotase from Aquifex aeolicus at 1.7A resolution
J. Mol. Biol.
348
535-547
2005
Aquifex aeolicus, Aquifex aeolicus (O66990)
brenda
Robles Lopez, S.M.; Hortua Triana, M.A.; Zimmermann, B.H.
Cloning and preliminary characterization of the dihydroorotase from Toxoplasma gondii
Mol. Biochem. Parasitol.
148
93-98
2006
Arabidopsis thaliana (O04904), Escherichia coli (P05020), Homo sapiens (P27708), Mesocricetus auratus (P08955), Mycobacterium leprae, Plasmodium berghei (Q4YUX2), Saccharomyces cerevisiae, Toxoplasma gondii (Q8MXA5), Toxoplasma gondii, Trypanosoma cruzi (O76138), Ustilago maydis (P31301)
brenda
Chen, K.F.; Lai, Y.Y.; Sun, H.S.; Tsai, S.J.
Transcriptional repression of human cad gene by hypoxia inducible factor-1alpha
Nucleic Acids Res.
33
5190-5198
2005
Homo sapiens
brenda
Liang, Y.H.; Liu, X.; Wang, J.; Li, L.; Su, X.D.
Protein preperation, crystallization and preliminary X-ray crystallographic studies of dihydroorotase from Bacillus subtilis
Protein Pept. Lett.
12
717-719
2005
Bacillus subtilis
brenda
Lee, M.; Maher, M.J.; Guss, J.M.
Structure of the T109S mutant of Escherichia coli dihydroorotase complexed with the inhibitor 5-fluoroorotate: catalytic activity is reflected by the crystal form
Acta Crystallogr. Sect. F
F63
154-161
2007
Escherichia coli, Escherichia coli (P05020)
brenda
Krungkrai, S.R.; Wutipraditkul, N.; Krungkrai, J.
Dihydroorotase of human malarial parasite Plasmodium falciparum differs from host enzyme
Biochem. Biophys. Res. Commun.
366
821-826
2008
Plasmodium falciparum
brenda
Lee, M.; Maher, M.J.; Christopherson, R.I.; Guss, J.M.
Kinetic and structural analysis of mutant Escherichia coli dihydroorotases: a flexible loop stabilizes the transition state
Biochemistry
46
10538-10550
2007
Escherichia coli, Escherichia coli (P05020)
brenda
Lee, M.; Chan, C.W.; Graham, S.C.; Christopherson, R.I.; Guss, J.M.; Maher, M.J.
Structures of ligand-free and inhibitor complexes of dihydroorotase from Escherichia coli: implications for loop movement in inhibitor design
J. Mol. Biol.
370
812-825
2007
Escherichia coli, Escherichia coli (P05020)
brenda
Zhang, P.; Martin, P.D.; Purcarea, C.; Vaishnav, A.; Brunzelle, J.S.; Fernando, R.; Guy-Evans, H.I.; Evans, D.R.; Edwards, B.F.
Dihydroorotase from the hyperthermophile Aquifex aeolicus is activated by stoichiometric association with aspartate transcarbamoylase and forms a one-pot reactor for pyrimidine biosynthesis
Biochemistry
48
766-778
2009
Aquifex aeolicus, Aquifex aeolicus (O66990)
brenda
Schroeder, P.E.; Patel, D.; Hasinoff, B.B.
The dihydroorotase inhibitor 5-aminoorotic acid inhibits the metabolism in the rat of the cardioprotective drug dexrazoxane and its one-ring open metabolites
Drug Metab. Dispos.
36
1780-1785
2008
Rattus norvegicus
brenda
Adama, C.; Bernard, S.; Brice, B.; Felix, Y.; Joseph, D.
Optimization of biomass and dihydroorotase (DHOase) production by Saccharomyces cerevisiae MNJ3 (pMNJ1)
Afr. J. Biotechnol.
8
037-041
2009
Saccharomyces cerevisiae
-
brenda
West, T.P.
Effect of carbon source on pyrimidine biosynthesis in Pseudomonas oryzihabitans
J. Basic Microbiol.
50
397-400
2010
Pseudomonas oryzihabitans
brenda
Wang, C.C.; Tsau, H.W.; Chen, W.T.; Huang, C.Y.
Identification and characterization of a putative dihydroorotase, KPN01074, from Klebsiella pneumoniae
Protein J.
29
445-452
2010
Klebsiella pneumoniae (A6T7D6), Klebsiella pneumoniae
brenda
Mehboob, S.; Mulhearn, D.C.; Truong, K.; Johnson, M.E.; Santarsiero, B.D.
Structure of dihydroorotase from Bacillus anthracis at 2.6 A resolution
Acta Crystallogr. Sect. F
66
1432-1435
2010
Bacillus anthracis, Bacillus anthracis (Q81WF0)
brenda
Nara, T.; Hashimoto, M.; Hirawake, H.; Liao, C.W.; Fukai, Y.; Suzuki, S.; Tsubouchi, A.; Morales, J.; Takamiya, S.; Fujimura, T.; Taka, H.; Mineki, R.; Fan, C.K.; Inaoka, D.K.; Inoue, M.; Tanaka, A.; Harada, S.; Kita, K.; Aoki, T.
Molecular interaction of the first 3 enzymes of the de novo pyrimidine biosynthetic pathway of Trypanosoma cruzi
Biochem. Biophys. Res. Commun.
418
140-143
2012
Trypanosoma cruzi (O76138), Trypanosoma cruzi
brenda
Peng, W.F.; Huang, C.Y.
Allantoinase and dihydroorotase binding and inhibition by flavonols and the substrates of cyclic amidohydrolases
Biochimie
101
113-122
2014
Klebsiella pneumoniae
brenda
Truong, L.; Hevener, K.E.; Rice, A.J.; Patel, K.; Johnson, M.E.; Lee, H.
High-level expression, purification, and characterization of Staphylococcus aureus dihydroorotase (PyrC) as a cleavable His-SUMO fusion
Protein Expr. Purif.
88
98-106
2013
Staphylococcus aureus (P65907), Staphylococcus aureus, Staphylococcus aureus MW2 (P65907)
brenda
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