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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
- carbamoyl-phosphate
- phosphoribosyltransferase
- atcase
- cpsase
-
glutamine-dependent
- l-dihydroorotate
-
2.1.3.2
- orotidine
-
carbamoyltransferase
- n-phosphonacetyl-l-aspartate
-
pyre
- allantoinase
- hydantoinase
- dihydro-ouabain
- dihydropyrimidinase
-
n-carbamyl-l-aspartate
- drug development
-
amidohydrolases
- imidase
- medicine
- synthesis
-
6.3.5.5
-
succinate-grown
- 5-fluoroorotate
The enzyme appears in viruses and cellular organisms
Synonyms
amidohydrolase family protein, BcDHOase, CAD, carbamoylaspartic dehydrase, Class I DHOase, DHO, DHOase, dihydroorotase, dihydroorotase domain, dihydroorotate dehydrolase, 
more
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SYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
amidohydrolase family protein
carbamoylaspartic dehydrase
-
-
-
-
Class I DHOase
DHO
DHOase
dihydroorotase
dihydroorotate dehydrolase
-
-
-
-
hDHOase
LdDHOase
pyrC
type I DHOase
VcDHO
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
complex formation of enzyme with aspartate transcarbamoylase drives reaction in the biosynthetic direction
-
(S)-dihydroorotate + H2O = N-carbamoyl-L-aspartate
unique catalytic mechanism
-
(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,Nâ-[(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
-
-
?
(S)-dihydroorotate + H2O
N-carbamoyl-L-aspartate
Methanocaldococcus jannaschii ATCC 624773
-
-
-
-
r
(S)-dihydroorotate + H2O
N-carbamoyl-L-aspartate
-
-
-
r
(S)-dihydroorotate + H2O
N-carbamoyl-L-aspartate
-
-
-
r
L-carbamoylaspartate
L-dihydroorotate + H2O
-
-
-
-
r
N-carbamoyl-L-aspartate
(S)-dihydroorotate + H2O
preferred reaction direction
-
-
r
N-carbamoyl-L-aspartate
(S)-dihydroorotate + H2O
Leishmania donovani BHU 1081
preferred reaction direction
-
-
r
N-carbamoyl-L-aspartate
(S)-dihydroorotate + H2O
Methanocaldococcus jannaschii ATCC 624773
-
-
-
-
r
N-carbamoyl-L-aspartate
(S)-dihydroorotate + H2O
-
-
-
r
N-carbamoyl-L-aspartate
(S)-dihydroorotate + H2O
-
-
-
r
N-carbamoyl-L-aspartate
L-dihydroorotate + H2O
-
-
-
-
r
additional information
?
-
the DHOase reaction is reversible and pH-dependent
-
-
-
additional information
?
-
-
the DHOase reaction is reversible and pH-dependent
-
-
-
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
-
-
?
additional information
?
-
N-carbamoyl-L-aspartate is preferred as the physiological substrate
-
-
-
additional information
?
-
malate can bind to the active site
-
-
-
additional information
?
-
N-carbamoyl-L-aspartate is preferred as the physiological substrate
-
-
-
additional information
?
-
malate can bind to the active site
-
-
-
additional information
?
-
N-carbamoyl-L-aspartate is preferred as the physiological substrate
-
-
-
additional information
?
-
malate can bind to the active site
-
-
-
additional information
?
-
N-carbamoyl-L-aspartate is preferred as the physiological substrate
-
-
-
additional information
?
-
-
N-carbamoyl-L-aspartate is preferred as the physiological substrate
-
-
-
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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
(S)-dihydroorotate + H2O
N-carbamoyl-L-aspartate
Methanocaldococcus jannaschii ATCC 624773
-
-
-
-
r
(S)-dihydroorotate + H2O
N-carbamoyl-L-aspartate
-
-
-
r
(S)-dihydroorotate + H2O
N-carbamoyl-L-aspartate
-
-
-
r
N-carbamoyl-L-aspartate
(S)-dihydroorotate + H2O
Methanocaldococcus jannaschii ATCC 624773
-
-
-
-
r
N-carbamoyl-L-aspartate
(S)-dihydroorotate + H2O
-
-
-
r
N-carbamoyl-L-aspartate
(S)-dihydroorotate + H2O
-
-
-
r
N-carbamoyl-L-aspartate
L-dihydroorotate + H2O
-
-
-
-
r
additional information
?
-
N-carbamoyl-L-aspartate is preferred as the physiological substrate
-
-
-
additional information
?
-
N-carbamoyl-L-aspartate is preferred as the physiological substrate
-
-
-
additional information
?
-
N-carbamoyl-L-aspartate is preferred as the physiological substrate
-
-
-
additional information
?
-
N-carbamoyl-L-aspartate is preferred as the physiological substrate
-
-
-
additional information
?
-
-
N-carbamoyl-L-aspartate is preferred as the physiological substrate
-
-
-
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Zn2+
Zn2+
DHOase is a zinc-dependent metalloenzyme, analysis of the catalytic zinc environment, structure, overview. Cys181 is observed bonded to the zinc atom of the catalytic site, with residues 181-187 visible and tethered by the interaction of loop A with other regions of the protein. On one end, the 1200 bar zinc coordination is like in the monoclinic-ap form with the Cys181 recruited again by the zinc atom, instead of the water molecule; the Cys-181 sulfur atom rotates and becomes more and more closely connected to the zinc when pressure increases
Zn2+
two active site Zn2+ ions, Zn2+ ions coordinated by a carboxylated K102, while D250 abstracts the proton from the amide nitrogen of N-carbamoyl-L-aspartate
Zn2+
active sites contain Zn atoms coordinated by His and Asp residues
Zn2+
mono-zinc metalloenzyme, the active site of the huDHOase K1556A mutant contains one metal ion. Posttranslational carbamylated Lys is not required for Znalpha binding in huDHOase
Zn2+
-
a zinc-dependent metalloenzyme, average of 2.05 mol zinc per mol protein classifying the Methanocaldococcus jannaschii DHOase as a two-zinc center protein
Zn2+
dihydroorotase enzyme is a di-metalloenzyme that is dependent upon two zinc atoms in the active site for activity
Zn2+
the active site of VcDHO contains two zinc ions: Znalpha in trigonal bipyramidal coordination and Znbeta in tetrahedral coordination. Each zinc ion is coordinated by two histidine residues, His13 and His15 for Znalpha, and His135 and His173 for Znbeta in VcDHO, structure analysis, overview
Zn2+
the active site of YpDHO contains two zinc ions: Znalpha in trigonal bipyramidal coordination and Znbeta in tetrahedral coordination. Each zinc ion is coordinated by two histidine residues, His17 and His19 for Znalpha, and His140 and His178 for Znbeta in YpDHO, structure analysis, overview
Zn2+
-
tagged enzyme contains ca. 1.0 zinc atom per subunit measured by atomic absorption spectroscopy
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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
1-(8-methoxy-1,3,4,5-tetrahydro-2H-pyrido[4,3-b]indol-2-yl)ethan-1-one
-
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
6'-methoxy-2',3',4',9'-tetrahydrospiro[oxane-4,1'-pyrido[3,4-b]indole]
-
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
DHCEDD
the peptide corresponds to the sequence 179DHCEDD185, and causes 50% inhibition of the wild-type enzyme
DHCEDDKLA
the peptide corresponds to the sequence 179DHCEDDKLA187, and causes 76% inhibition of the wild-type enzyme
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
[6-fluoro-1-(methylsulfanyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl]cyanamide
-
[6-methoxy-1-(methylsulfanyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl]cyanamide
-
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
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
kaempferol
kaempferol mediates perturbation of the pyrimidine pathway
additional information
pressure induces irreversible dissociation of the obligate ATC trimer, and as a consequence the DHO is also inactivated. Inhibition of DHO by small peptides that mimic the loop residues
-
additional information
-
pressure induces irreversible dissociation of the obligate ATC trimer, and as a consequence the DHO is also inactivated. Inhibition of DHO by small peptides that mimic the loop residues
-
additional information
inhibitor high-throughput screening, competitive inhibition, determination of the equilibrium dissociation constant determined by surface plasmon resonance with 1 mM N-carbamoyl-L-aspartate, overview
-
additional information
-
inhibitor high-throughput screening, competitive inhibition, determination of the equilibrium dissociation constant determined by surface plasmon resonance with 1 mM N-carbamoyl-L-aspartate, overview
-
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
-
additional information
malate can bind to the active site
-
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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
additional information
pressure-induced activation of dihydroorotase, overview
-
additional information
-
pressure-induced activation of dihydroorotase, overview
-
additional information
reversible activation of latent dihydroorotase from Aquifex aeolicus by moderate hydrostatic pressure. Moderate hydrostatic pressure applied to the isolated DHO subunit mimics the complex formation and reversibly activates the isolated subunit in the absence of ATC, suggesting that the loop has been displaced from the active site. This effect of pressure is explained by the negative volume change associated with the disruption of ionic interactions and exposure of ionized amino acids to the solvent (electrostriction). The isolated DHO protein, a 45-kDa monomer, lacks catalytic activity but becomes active upon formation of a dodecameric complex with aspartate transcarbamoylase (ATC, EC 2.1.3.2)
-
additional information
-
reversible activation of latent dihydroorotase from Aquifex aeolicus by moderate hydrostatic pressure. Moderate hydrostatic pressure applied to the isolated DHO subunit mimics the complex formation and reversibly activates the isolated subunit in the absence of ATC, suggesting that the loop has been displaced from the active site. This effect of pressure is explained by the negative volume change associated with the disruption of ionic interactions and exposure of ionized amino acids to the solvent (electrostriction). The isolated DHO protein, a 45-kDa monomer, lacks catalytic activity but becomes active upon formation of a dodecameric complex with aspartate transcarbamoylase (ATC, EC 2.1.3.2)
-
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
-
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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.
Maternal Death
No one data source captures all: A nested case-control study of the completeness of maternal death reporting in Banten Province, Indonesia.
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.
Uterine Cervical Neoplasms
Dihydroouabain, a novel radiosensitizer for cervical cancer identified by automated high-throughput screening.
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.114
(S)-dihydroorotate
pH 8.3, temperature not specified in the publication, recombinant wild-type enzyme
0.118
(S)-dihydroorotate
pH 8.3, 25°C, recombinant detagged enzyme
0.167
(S)-dihydroorotate
pH 8.3, 25°C, recombinant tagged enzyme
0.323
N-(aminocarbonyl)-L-aspartic acid
purified recombinant enzyme, at 37°C
0.112
N-carbamoyl-L-aspartate
pH 5.8, temperature not specified in the publication, recombinant wild-type enzyme
0.24
N-carbamoyl-L-aspartate
pH 8.0, 74°C, recombinant DHO-ATC complex with mutant DHO E179V
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
0.47
N-carbamoyl-L-aspartate
pH 8.0, 74°C, recombinant DHO-ATC complex with mutant DHO E183V
1.183
N-carbamoyl-L-aspartate
pH 5.8, temperature not specified in the publication, recombinant mutant N93A
2.38
N-carbamoyl-L-aspartate
pH 8.0, 74°C, recombinant wild-type DHO-ATC complex
3.89
N-carbamoyl-L-aspartate
pH 8.0, 74°C, recombinant DHO-ATC complex with mutant DHO C181A
6.2
N-carbamoyl-L-aspartate
pH 8.0, 74°C, recombinant isolated DHO mutant DHO E179V
24
N-carbamoyl-L-aspartate
pH 8.0, 74°C, recombinant isolated DHO mutant DHO E183V
additional information
carbamoyl aspartate
mutants D250A, D250H, D250N, R20Q, R20M, N44A, H254N inactive
additional information
additional information
-
Michaelis-Menten kinetics
-
additional information
additional information
Michaelis-Menten kinetics
-
additional information
additional information
-
Michaelis-Menten kinetics
-
additional information
additional information
Michaelis-Menten kinetics
-
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
-
additional information
additional information
-
forward and reverse kinetic rate constants for both tagged and wild-type enzyme, kinetic analysis, overview
-
additional information
additional information
-
Michaelis-Menten kinetic analysis, recombinant enzyme
-
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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
1.87
(S)-dihydroorotate
pH 8.3, 25°C, recombinant tagged enzyme
1.9
(S)-dihydroorotate
pH 8.3, temperature not specified in the publication, recombinant wild-type enzyme
11.5
N-(aminocarbonyl)-L-aspartic acid
mutant enzyme T110A, at pH 5.8
49.8
N-(aminocarbonyl)-L-aspartic acid
mutant enzyme T109S, at pH 5.8
59.7
N-(aminocarbonyl)-L-aspartic acid
mutant enzyme T110S, at pH 5.8
145.5
N-(aminocarbonyl)-L-aspartic acid
mutant enzyme T110V, at pH 5.8
1.4
N-carbamoyl-L-aspartate
pH 5.8, temperature not specified in the publication, recombinant mutant N93A
1.9
N-carbamoyl-L-aspartate
pH 8.3, 25°C, recombinant tagged enzyme
2.1
N-carbamoyl-L-aspartate
pH 5.8, temperature not specified in the publication, recombinant wild-type enzyme
2.48
N-carbamoyl-L-aspartate
pH 8.3, 25°C, recombinant detagged enzyme
3
N-carbamoyl-L-aspartate
pH 8.0, 74°C, recombinant isolated DHO mutant DHO E179V
6.7
N-carbamoyl-L-aspartate
pH 8.0, 74°C, recombinant isolated DHO mutant DHO E183V
9.2
N-carbamoyl-L-aspartate
pH 8.0, 74°C, recombinant DHO-ATC complex with mutant DHO E179V
9.7
N-carbamoyl-L-aspartate
pH 8.0, 74°C, recombinant DHO-ATC complex with mutant DHO E183V
15.5
N-carbamoyl-L-aspartate
pH 8.0, 74°C, recombinant DHO-ATC complex with mutant DHO C181A
62.3
N-carbamoyl-L-aspartate
pH 8.0, 74°C, recombinant wild-type DHO-ATC complex
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
11.2
(S)-dihydroorotate
pH 8.3, 25°C, recombinant tagged enzyme
13.1
(S)-dihydroorotate
pH 8.3, 25°C, recombinant detagged enzyme
16.67
(S)-dihydroorotate
pH 8.3, temperature not specified in the publication, recombinant wild-type enzyme
0.28
N-carbamoyl-L-aspartate
pH 8.0, 74°C, recombinant isolated DHO mutant DHO E183V
0.48
N-carbamoyl-L-aspartate
pH 8.0, 74°C, recombinant isolated DHO mutant DHO E179V
1.18
N-carbamoyl-L-aspartate
pH 5.8, temperature not specified in the publication, recombinant mutant N93A
3.98
N-carbamoyl-L-aspartate
pH 8.0, 74°C, recombinant DHO-ATC complex with mutant DHO C181A
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
18.75
N-carbamoyl-L-aspartate
pH 5.8, temperature not specified in the publication, recombinant wild-type enzyme
20.6
N-carbamoyl-L-aspartate
pH 8.0, 74°C, recombinant DHO-ATC complex with mutant DHO E183V
26.2
N-carbamoyl-L-aspartate
pH 8.0, 74°C, recombinant wild-type DHO-ATC complex
38.3
N-carbamoyl-L-aspartate
pH 8.0, 74°C, recombinant DHO-ATC complex with mutant DHO E179V
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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
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
5.8
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pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
25
37
70
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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
in noncovalent association with aspartate transcarbamoylase, possible model for the mammalian polypeptide chain CPSase/ATCase/DHOase during pyrimidine biosynthesis
UniProt
brenda
strain RH lambda Zap II cDNA library, strain PC117 and 5TN
SwissProt
brenda
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SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
additional information
maintained by serial passage in the peritoneal cavity of CFW mice
brenda
additional information
-
optimal growth of the organism of 85°C
brenda
additional information
Methanocaldococcus jannaschii ATCC 624773
-
optimal growth of the organism of 85°C
-
brenda
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LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
malfunction
metabolism
physiological function
additional information
evolution
despite evolutionary divergence, the CAD DHOase active site components are highly conserved with those in bacterial DHOases, encoded as monofunctional enzymes. An important element for catalysis, conserved from Escherichia coli to humans, is a flexible loop that closes as a lid over the active site. The flexible loop exhibits a two-amino acid signature that is characteristic for each DHOase type
evolution
dihydroorotase (DHO) is an amidohydrolase that catalyzes the reversible condensation of carbamoyl aspartate to form dihydroorotate in de novo pyrimidine biosynthesis in virtually all organisms. Although the same reaction is catalyzed by all DHOs, the structure, oligomeric organization, and metal content of this family of enzymes is diverse
evolution
dihydroorotase (DHOase) is a member of the cyclic amidohydrolase family, which also includes allantoinase, hydantoinase, dihydropyrimidinase (DHPase), and imidase. Almost all of these zinc metalloenzymes possess a binuclear metal center in which two metal ions are bridged by a post-translational carbamylated Lys
evolution
mammals have a large, multifunctional dihydroorotate synthetase (CAD) enzyme for the first three steps of the de novo pyrimidine biosynthesis pathway, while prokaryotes use three separate monofunctional enzymes for each. DHOase is divided into two evolutionary classes, with very low sequence identity (< 30%) between classes. Class I DHOases are found in gram-positive bacteria, mold, and insects, while Class II DHOases are found in gram-negative bacteria and fungi. The major structural difference between the Class I and Class II DHOase is the longer catalytic loop of Class II counterparts
evolution
the enzyme belongs to the amidohydrolase superfamily
evolution
the structure of YpDHO has essentially the same conformation as the structure of DHO from Escherichia coli (EcDHO), the most thoroughly studied bacterial type II DHO. The enzyme belongs to the amidohydrolase superfamily
evolution
-
mammals have a large, multifunctional dihydroorotate synthetase (CAD) enzyme for the first three steps of the de novo pyrimidine biosynthesis pathway, while prokaryotes use three separate monofunctional enzymes for each. DHOase is divided into two evolutionary classes, with very low sequence identity (< 30%) between classes. Class I DHOases are found in gram-positive bacteria, mold, and insects, while Class II DHOases are found in gram-negative bacteria and fungi. The major structural difference between the Class I and Class II DHOase is the longer catalytic loop of Class II counterparts
-
evolution
-
the enzyme belongs to the amidohydrolase superfamily
-
evolution
-
the enzyme belongs to the amidohydrolase superfamily
-
malfunction
a huDHOase chimera bearing the Escherichia coli DHOase flexible loop is inactive, suggesting the presence of distinctive elements in the flexible loop of huDHOase that cannot be replaced by the bacterial sequence. Substitutions of Phe1563 with Ala, Leu, or Thr prevent the closure of the flexible loop and inactivated the protein, whereas substitution with Tyr enhances the interactions of the loop in the closed position and reduced fluctuations and the reaction rate
malfunction
a parallel salvage pathway of pyrimidine biosynthesis exists, since kaempferol cannot completely inhibit the pathway in vivo
malfunction
the replacement of the zinc ligand Cys181 with glycine does not restore the latent catalytic activity suggesting that it plays a minor role in stabilizing loop A
malfunction
Leishmania donovani BHU 1081
-
a parallel salvage pathway of pyrimidine biosynthesis exists, since kaempferol cannot completely inhibit the pathway in vivo
-
metabolism
third enzyme in the bacterial de novo pyrimidine biosynthesis pathway, overview
metabolism
-
catalyzes the conversion of N-carbamoyl-L-aspartate to dihydroorotate in the third step of the de novo biosynthesis of pyrimidines
metabolism
de novo pyrimidine biosynthesis pathway is well developed and functional in protozoan parasite Leishmania donovani. The dihydroorotase (LdDHOase) is the third enzyme of the pathway
metabolism
dihydroorotase (DHO) is an amidohydrolase that catalyzes the reversible condensation of carbamoyl aspartate to form dihydroorotate in de novo pyrimidine biosynthesis
metabolism
dihydroorotase (DHOase) catalyses the third reaction of the de novo pyrimidine biosynthetic pathway, the reversible condensation of carbamylaspartate into dihydroorotate
metabolism
the de novo pyrimidine biosynthesis pathway is essential for the proliferation of many pathogens. One of the pathway enzymes, dihydroorotase (DHO), catalyzes the reversible interconversion of N-carbamoyl-L-aspartate to 4,5-dihydroorotate. De novo pyrimidine biosynthesis pathway, overview
metabolism
the de novo pyrimidine biosynthesis pathway is essential for the proliferation of many pathogens. One of the pathway enzymes, dihydroorotase (DHO), catalyzes the reversible interconversion of N-carbamoyl-L-aspartate to 4,5-dihydroorotate. De novo pyrimidine biosynthesis pathway, overview
metabolism
the dihydroorotase (DHOase) domain of the multifunctional protein carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase (CAD) catalyzes the third step in the de novo biosynthesis of pyrimidine nucleotides in animals
metabolism
Leishmania donovani BHU 1081
-
de novo pyrimidine biosynthesis pathway is well developed and functional in protozoan parasite Leishmania donovani. The dihydroorotase (LdDHOase) is the third enzyme of the pathway
-
metabolism
-
the de novo pyrimidine biosynthesis pathway is essential for the proliferation of many pathogens. One of the pathway enzymes, dihydroorotase (DHO), catalyzes the reversible interconversion of N-carbamoyl-L-aspartate to 4,5-dihydroorotate. De novo pyrimidine biosynthesis pathway, overview
-
metabolism
Methanocaldococcus jannaschii ATCC 624773
-
catalyzes the conversion of N-carbamoyl-L-aspartate to dihydroorotate in the third step of the de novo biosynthesis of pyrimidines
-
metabolism
-
third enzyme in the bacterial de novo pyrimidine biosynthesis pathway, overview
-
metabolism
-
the de novo pyrimidine biosynthesis pathway is essential for the proliferation of many pathogens. One of the pathway enzymes, dihydroorotase (DHO), catalyzes the reversible interconversion of N-carbamoyl-L-aspartate to 4,5-dihydroorotate. De novo pyrimidine biosynthesis pathway, overview
-
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
physiological function
cells rely on nucleotide synthesis for survival and proliferation, and pyrimidines are essential building blocks of RNA and DNA. Dihydroorotase (DHOase), the third enzyme in the essential de novo pyrimidine pathway, is responsible for the reversible cyclization of carbamyl-asparate (Ca-asp) to dihydroorotate (DHO)
physiological function
the enzyme catalyzes the reversible cyclization of N-carbamyl aspartate to dihydroorotate
physiological function
Leishmania donovani BHU 1081
-
the enzyme catalyzes the reversible cyclization of N-carbamyl aspartate to dihydroorotate
-
physiological function
-
cells rely on nucleotide synthesis for survival and proliferation, and pyrimidines are essential building blocks of RNA and DNA. Dihydroorotase (DHOase), the third enzyme in the essential de novo pyrimidine pathway, is responsible for the reversible cyclization of carbamyl-asparate (Ca-asp) to dihydroorotate (DHO)
-
additional information
-
the enzyme contains four histidine, one aspartate, and one post-carboxylated lysine residue, which are required for metal binding and catalytic activity
additional information
analysis of the catalytic flexible loop in the dihydroorotase domain of the human multi-enzymatic protein CAD, molecular dynamics simulations, overview. Residue Phe1563, a residue absolutely conserved at the tip of the flexible loop in CAD's DHOase domain, is a critical element for the conformational equilibrium between the two catalytic states of the protein. Key role of Phe-1563 in configuring the active site and in promoting substrate strain and catalysis. The flexible loop reaches in toward the active site with N-carbamoyl-L-aspartate bound and is proposed to aid in catalysis by orienting and increasing the electrophilicity of the substrate, excluding water molecules, and stabilizing the transition-state. Then, upon the formation of DHO, the loop moves away from the active site, facilitating product release. As an exception, bacterial type I DHOases present a rigid and shorter loop that interacts minimally with the substrate, requiring the intimate association with ATCase to complete the active site and attain full activity
additional information
-
analysis of the catalytic flexible loop in the dihydroorotase domain of the human multi-enzymatic protein CAD, molecular dynamics simulations, overview. Residue Phe1563, a residue absolutely conserved at the tip of the flexible loop in CAD's DHOase domain, is a critical element for the conformational equilibrium between the two catalytic states of the protein. Key role of Phe-1563 in configuring the active site and in promoting substrate strain and catalysis. The flexible loop reaches in toward the active site with N-carbamoyl-L-aspartate bound and is proposed to aid in catalysis by orienting and increasing the electrophilicity of the substrate, excluding water molecules, and stabilizing the transition-state. Then, upon the formation of DHO, the loop moves away from the active site, facilitating product release. As an exception, bacterial type I DHOases present a rigid and shorter loop that interacts minimally with the substrate, requiring the intimate association with ATCase to complete the active site and attain full activity
additional information
homology modeling of LdDHOase showing the active site residues, overview
additional information
-
homology modeling of LdDHOase showing the active site residues, overview
additional information
in the hyperthermophilic bacterium Aquifex aeolicus, aspartate transcarbamylase (ATCase, EC 2.1.3.2) and dihydroorotase (DHOase) are noncovalently associated. Upon dissociation, ATCase keeps its activity entirely while DHOase is totally inactivated. High pressure fully restores the activity of this isolated DHOase. Under high-hydrostatic pressure, at 600 bar, and to a greater extent at 1200 bar, the orthorhombic form of DHOase displays a structure, which includes the Cys-181 bridge of the C2-ap form and some additional residues of the missing loops that become ordered and visible in the electron density
additional information
-
in the hyperthermophilic bacterium Aquifex aeolicus, aspartate transcarbamylase (ATCase, EC 2.1.3.2) and dihydroorotase (DHOase) are noncovalently associated. Upon dissociation, ATCase keeps its activity entirely while DHOase is totally inactivated. High pressure fully restores the activity of this isolated DHOase. Under high-hydrostatic pressure, at 600 bar, and to a greater extent at 1200 bar, the orthorhombic form of DHOase displays a structure, which includes the Cys-181 bridge of the C2-ap form and some additional residues of the missing loops that become ordered and visible in the electron density
additional information
structure homology modelling using the Plasmodium falciparum enzyme structure as model (PfDHO, UniProtKB ID Q8IKA9)
additional information
-
structure homology modelling using the Plasmodium falciparum enzyme structure as model (PfDHO, UniProtKB ID Q8IKA9)
additional information
structure homology modelling using the Plasmodium falciparum enzyme structure as model (PfDHO,UniProtKB ID Q8IKA9)
additional information
-
structure homology modelling using the structure of DHOase from Methanocaldococcus jannaschii strain ATCC 43067 / DSM 2661 / JAL-1 / JCM 10045 / NBRC 100440, UniProt ID Q58885. Sequence and structure comparisons
additional information
the isolated DHO protein, a 45-kDa monomer, lacks catalytic activity but becomes active upon formation of a dodecameric complex with aspartate transcarbamoylase (ATC, EC 2.1.3.2). In the isolated DHO, a flexible loop occludes the active site blocking the access of substrates. The loop is mostly disordered but is tethered to the active site region by several electrostatic and hydrogen bonds. This loop becomes ordered and is displaced from the active site upon formation of DHO-ATC complex. The application of pressure to the complex causes its time-dependent dissociation and the loss of both DHO and ATC activities. Pressure induces irreversible dissociation of the obligate ATC trimer, and as a consequence the DHO is also inactivated
additional information
-
the isolated DHO protein, a 45-kDa monomer, lacks catalytic activity but becomes active upon formation of a dodecameric complex with aspartate transcarbamoylase (ATC, EC 2.1.3.2). In the isolated DHO, a flexible loop occludes the active site blocking the access of substrates. The loop is mostly disordered but is tethered to the active site region by several electrostatic and hydrogen bonds. This loop becomes ordered and is displaced from the active site upon formation of DHO-ATC complex. The application of pressure to the complex causes its time-dependent dissociation and the loss of both DHO and ATC activities. Pressure induces irreversible dissociation of the obligate ATC trimer, and as a consequence the DHO is also inactivated
additional information
the two Zn2+ ions hold the substrate Ca-asp in position along with the active site residues, Arg60, Asn93, and His308, which also overlap well, suggesting the substrate is stabilized by the same hydrogen bond interactions. The hydrogen bond interaction between Ca-asp and the two threonine on the catalytic loop play a crucial role in Class II enzymes, but neither of the threonine residues is present in Bacillus anthracis DHOase, structure analysis reveals that glycine (G152) in the shorter Class I loop serves this function. Catalytic mechanism, overview
additional information
-
the two Zn2+ ions hold the substrate Ca-asp in position along with the active site residues, Arg60, Asn93, and His308, which also overlap well, suggesting the substrate is stabilized by the same hydrogen bond interactions. The hydrogen bond interaction between Ca-asp and the two threonine on the catalytic loop play a crucial role in Class II enzymes, but neither of the threonine residues is present in Bacillus anthracis DHOase, structure analysis reveals that glycine (G152) in the shorter Class I loop serves this function. Catalytic mechanism, overview
additional information
Leishmania donovani BHU 1081
-
homology modeling of LdDHOase showing the active site residues, overview
-
additional information
-
the two Zn2+ ions hold the substrate Ca-asp in position along with the active site residues, Arg60, Asn93, and His308, which also overlap well, suggesting the substrate is stabilized by the same hydrogen bond interactions. The hydrogen bond interaction between Ca-asp and the two threonine on the catalytic loop play a crucial role in Class II enzymes, but neither of the threonine residues is present in Bacillus anthracis DHOase, structure analysis reveals that glycine (G152) in the shorter Class I loop serves this function. Catalytic mechanism, overview
-
additional information
-
structure homology modelling using the Plasmodium falciparum enzyme structure as model (PfDHO,UniProtKB ID Q8IKA9)
-
additional information
Methanocaldococcus jannaschii ATCC 624773
-
structure homology modelling using the structure of DHOase from Methanocaldococcus jannaschii strain ATCC 43067 / DSM 2661 / JAL-1 / JCM 10045 / NBRC 100440, UniProt ID Q58885. Sequence and structure comparisons
-
additional information
-
structure homology modelling using the Plasmodium falciparum enzyme structure as model (PfDHO,UniProtKB ID Q8IKA9)
-
Show AA Sequence and Transmembrane Helices (45570 entries)
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PDB
SCOP
CATH
UNIPROT
ORGANISM
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
38000
40000
42000
45600
48000
48090
49000
498000
-
gel filtration, DHOase in complex with aspartate transcarbamoylase
500000
-
aspartate transcarbamoylase-dihydroorotase complex, gel filtration
48090
-
electrospray ionization mass spectroscopy, enzyme with hexahistidine tag
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
?
x * 43900, about, sequence calculation, x* 48000, recombinant His6-tagged enzyme, SDS-PAGE
?
Leishmania donovani BHU 1081
-
x * 43900, about, sequence calculation, x* 48000, recombinant His6-tagged enzyme, SDS-PAGE
-
?
-
x * 47503-48104, sequence calculation, mass spectrometry, and SDS-PAGE
?
Methanocaldococcus jannaschii ATCC 624773
-
x * 47503-48104, sequence calculation, mass spectrometry, and SDS-PAGE
-
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
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
enzymes DHO-ATC complex structure, analysis of the quaternary structural organization and interactions between the subunits in the Aquifex aeolicus complex, overview
additional information
-
enzymes DHO-ATC complex structure, analysis of the quaternary structural organization and interactions between the subunits in the Aquifex aeolicus complex, overview
additional information
replacement of the flexible loop weakens the dimerization of huDHOase
additional information
-
replacement of the flexible loop weakens the dimerization of huDHOase
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
-
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
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
additional information
three-dimensional structure of DHO from Vibrio cholerae, structure analysis and comparison, overview
additional information
-
three-dimensional structure of DHO from Vibrio cholerae, structure analysis and comparison, overview
-
additional information
-
three-dimensional structure of DHO from Vibrio cholerae, structure analysis and comparison, overview
-
additional information
three-dimensional structure of DHO from Yersinia pestis, structure analysis and comparison, overview. Each subunit has a structure based on the (beta/alpha)8-barrel (TIM-barrel) fold, a characteristic of the amidohydrolase superfamily
additional information
-
three-dimensional structure of DHO from Yersinia pestis, structure analysis and comparison, overview. Each subunit has a structure based on the (beta/alpha)8-barrel (TIM-barrel) fold, a characteristic of the amidohydrolase superfamily
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POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
additional information
VcDHO contains a carboxylated lysine (Kcx) residue in position 96 and two S-oxy cysteine (Csx) residues in positions 259 and 261
additional information
-
VcDHO contains a carboxylated lysine (Kcx) residue in position 96 and two S-oxy cysteine (Csx) residues in positions 259 and 261
-
additional information
-
VcDHO contains a carboxylated lysine (Kcx) residue in position 96 and two S-oxy cysteine (Csx) residues in positions 259 and 261
-
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
crystal structure analysis of the zinc environment at ambient pressure, at 600 bar, and at 1200 bar
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
purified recombinant detagged wild-type enzyme complexed with N-carbamoyl-L-aspartate or (S)-dihydroorotate, hanging drop vapor diffusion method, mixing of 10 mg/ml protein solution, containing 1.5 mM ligand, with reservoir solution containing 0.1 M Bis-Tris, pH 5.5, 0.2 M NaCl, and 20% PEG 3350, X-ray diffraction structure determination and analysis at 2.45 A resolution, chains A and B are in one asymmetric unit. The active site of each monomer, and both substrates have relatively weak electron density due to low substrate occupancy
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
human DHOase domain K1556A mutant, hanging drop vapor diffusion method, mixing of 20 mg/ml protein in 20 mM HEPES and 100 mM NaCl, pH 7.0, with reservoir solution containing 2 M sodium chloride, 100 mM MES, 200 mM sodium acetate, pH 6.5, at room temperature, X-ray diffraction structure determination and analysis at 2.77 A resolution
several F1563 mutant variants of huDHOase bound to dihydroorotate, X-ray diffraction structure determination and analysis at 1.46-2.12 A resolution
purified recombinant His-tagged enzyme, enzyme proteolysis before crystallization, sitting drop vapor diffusion technique, mixing of 0.001 ml of 14 mg/ml protein in 20 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, 0.1% sodium azide, 0.5 mM TCEP, and and 20 mM L-citrulline, with 0.0015 ml of crystallization solution containing 133 mM sodium acetate, 67 mM sodium cacodylate-HCl, pH 6.5, 20% w/v PEG 8000, and 2.3% v/v 1-butanol, and equilibration against 0.034 ml of reservoir solution of 1.5 M NaCl, 4-7 days, 16°C, X-ray diffraction structure determination and analysis at 1.95 A resolution, molecular replacement, modelling
purified recombinant His-tagged enzyme, sitting drop vapor diffusion technique, mixing of 400 nl of 10 mg/ml protein in 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% glycerol, 0.1% sodium azide, and 0.5 mM TCEP, with 400 nl of crystallization solution containing 0.15 M DL-malic acid, pH 7.0, and 20% w/v PEG 3350, equilibration against 0.034 ml of reservoir solution of 1.3 M NaCl, 4-7 days, 16°C, X-ray diffraction structure determination and analysis at 2.4 A resolution, molecular replacement, modelling
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
C181A
site-directed mutagenesis, the mutant shows reduced activity and altered kinetics compared to wild-type
C181G
site-directed mutagenesis, the mutant does not restore the activity of DHOase that has been isolated from aspartate transcarbamylase (ATCase, EC 2.1.3.2)
D179V
site-directed mutagenesis, the mutant partially restores the activity of DHOase that has been isolated from aspartate transcarbamylase (ATCase, EC 2.1.3.2)
D183G
site-directed mutagenesis, the mutant partially restores the activity of DHOase that has been isolated from aspartate transcarbamylase (ATCase, EC 2.1.3.2)
E179V
site-directed mutagenesis, the mutant shows reduced activity and altered kinetics compared to wild-type
E183V
site-directed mutagenesis, the mutant shows reduced activity and altered kinetics compared to wild-type
N93A
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
N93A
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
-
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
F1563A
site-directed mutagenesis, the mutation prevents the closure of the flexible loop and inactivates the enzyme
F1563L
site-directed mutagenesis, the mutation prevents the closure of the flexible loop and inactivates the enzyme
F1563T
site-directed mutagenesis, the mutation prevents the closure of the flexible loop and inactivates the enzyme
F1563Y
site-directed mutagenesis, the mutation prevents enhances the interactions of the flexible loop in the closed position and reduces fluctuations and the reaction rate
K1556A
site-directed mutagenesis, the mutant shows no lysine carbamylation within the active site
additional information
additional information
in noncovalent association with aspartate transcarbamoylase, possible model for mammalian polypeptide chain CPSase/ATCase/DHOase during pyrimidine biosynthesis
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
additional information
-
deletion mutant DELTA107-116 DHOase shows negligible activity
additional information
construction of a huDHOase chimera bearing the Escherichia coli DHOase flexible loop, the mutant is inactive
additional information
-
construction of a huDHOase chimera bearing the Escherichia coli DHOase flexible loop, the mutant is inactive
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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
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
-
Ni-NTA agarose column chromatography, Poros Q column chromatography, and Sephadex G-75 gel filtration
-
recombinant enzyme from Escherichia coli by heat treatment at 85°C for 15 min, dialysis, cation exchange chromatography, and hydrophobic interaction chromatography
-
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and dialysis
-
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, desalting gel filtration, and ultrafiltration
recombinant His6-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) Gold by nickel affinity chromatography, tag cleavage through thrombin, dialysis, and gel filtration
recombinant isolated DHO and DDHO-ATC complex from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
recombinant mutant His-tagged enzyme 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
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, desalting gel filtration, and ultrafiltration
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, desalting gel filtration, and ultrafiltration
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli strain SO1263
expression in Escherichia coli
expression in Escherichia coli mutant strain SÃ1263 and strain X7014
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 DHO, recombinant expression of His6-tagged enzyme in Escherichia coli strain BL21(DE3), subcloning in Escherichia coli strain DH5alpha, quantitative real-time PCR expression analysis
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
gene pyrC, recombinant enzyme expression in Escherichia coli strain Rosetta-gami 2(DE3), subcloning in Escherichia coli strain DH5alpha
-
gene pyrC, recombinant expression of His6-tagged wildtype and mutant enzymes in Escherichia coli strain BL21(DE3) Gold
gene pyrC, recombinant expression of wild-type and mutant DHO enzymes in Escherichia coli strain BL21(DE3), coexpression with pyrB encoding with aspartate transcarbamoylase (ATC, EC 2.1.3.2)
gene pyrC, sequence comparisons, recombinant expression of His-tagged enzyme in Escherichia coli strain BL21(DE3)
recombinant overexpression of His-tagged enzyme in Escherichia coli strain BL21(DE3)
-
with a fusion peptide containing six histidine residues at the amino terminus
gene pyrC, sequence comparisons, recombinant expression of His-tagged enzyme in Escherichia coli strain BL21(DE3)
gene pyrC, sequence comparisons, recombinant expression of His-tagged enzyme in Escherichia coli strain BL21(DE3)
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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 substantial difference between bacterial and mammalian DHOs makes the bacterial enzyme a promising drug target for disrupting bacterial growth and thus an important candidate to evaluate as a response to antimicrobial resistance on a molecular level
drug development
the substantial difference between bacterial and mammalian DHOs makes the bacterial enzyme a promising drug target for disrupting bacterial growth and thus an important candidate to evaluate as a response to antimicrobial resistance on a molecular level
drug development
-
the substantial difference between bacterial and mammalian DHOs makes the bacterial enzyme a promising drug target for disrupting bacterial gr