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- Name help_outline (S)-dihydroorotate Identifier CHEBI:30864 Charge -1 Formula C5H5N2O4 InChIKeyhelp_outline UFIVEPVSAGBUSI-REOHCLBHSA-M SMILEShelp_outline [O-]C(=O)[C@@H]1CC(=O)NC(=O)N1 2D coordinates Mol file for the small molecule Search links Involved in 10 reaction(s) Find molecules that contain or resemble this structure Find proteins in UniProtKB for this molecule
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Name help_outline
a ubiquinone
Identifier
CHEBI:16389
(CAS: 1339-63-5)
help_outline
Charge
0
Formula
C9H10O4(C5H8)n
Search links
Involved in 49 reaction(s)
Find proteins in UniProtKB for this molecule
Form(s) in this reaction:
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Identifier: RHEA-COMP:9565Polymer name: a ubiquinonePolymerization index help_outline nFormula C9H10O4(C5H8)nCharge (0)(0)nMol File for the polymer
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Name help_outline
a ubiquinol
Identifier
CHEBI:17976
(CAS: 56275-39-9)
help_outline
Charge
0
Formula
C9H12O4(C5H8)n
Search links
Involved in 55 reaction(s)
Find proteins in UniProtKB for this molecule
Form(s) in this reaction:
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Identifier: RHEA-COMP:9566Polymer name: a ubiquinolPolymerization index help_outline nFormula C9H12O4(C5H8)nCharge (0)(0)nMol File for the polymer
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- Name help_outline orotate Identifier CHEBI:30839 (Beilstein: 3651747; CAS: 73-97-2) help_outline Charge -1 Formula C5H3N2O4 InChIKeyhelp_outline PXQPEWDEAKTCGB-UHFFFAOYSA-M SMILEShelp_outline [O-]C(=O)c1cc(=O)[nH]c(=O)[nH]1 2D coordinates Mol file for the small molecule Search links Involved in 14 reaction(s) Find molecules that contain or resemble this structure Find proteins in UniProtKB for this molecule
Cross-references
RHEA:28691 | RHEA:28692 | RHEA:28693 | RHEA:28694 | |
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Reaction direction help_outline | undefined | left-to-right | right-to-left | bidirectional |
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EcoCyc help_outline |
Related reactions help_outline
Specific form(s) of this reaction
More general form(s) of this reaction
Publications
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Mammalian dihydroorotate dehydrogenase: physical and catalytic properties of the primary enzyme.
Forman H.J., Kennedy J.
Arch Biochem Biophys 191:23-31(1978) [PubMed] [EuropePMC]
This publication is cited by 1 other entry.
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Evolutionary implications of the mosaic pyrimidine-biosynthetic pathway in eukaryotes.
Nara T., Hshimoto T., Aoki T.
The de-novo pyrimidine biosynthetic pathway involves six enzymes, in order from the first to the sixth step, carbamoyl-phosphate synthetase II (CPS II) comprising glutamine amidotransferase (GAT) and carbamoyl-phosphate synthetase (CPS) domains or subunits, aspartate carbamoyltransferase (ACT), di ... >> More
The de-novo pyrimidine biosynthetic pathway involves six enzymes, in order from the first to the sixth step, carbamoyl-phosphate synthetase II (CPS II) comprising glutamine amidotransferase (GAT) and carbamoyl-phosphate synthetase (CPS) domains or subunits, aspartate carbamoyltransferase (ACT), dihydroorotase (DHO), dihydroorotate dehydrogenase (DHOD), orotate phosphoribosyltransferase (OPRT), and orotidine-5'-monophosphate decarboxylase (OMPDC). In contrast with reports on molecular evolution of the individual enzymes, we attempted to draw an evolutionary picture of the whole pathway using the protein phylogeny. We demonstrate highly mosaic organizations of the pyrimidine biosynthetic pathway in eukaryotes. During evolution of the eukaryotic pathway, plants and fungi (or their ancestors) in particular may have secondarily acquired the characteristic enzymes. This is consistent with the fact that the organization of plant enzymes is highly chimeric: (1) two subunits of CPS II, GAT and CPS, cluster with a clade including cyanobacteria and red algal chloroplasts, (2) ACT not with a cyanobacterium, Synechocystis spp., irrespective of its putative signal sequence targeting into chloroplasts, and (3) DHO with a clade of proteobacteria. In fungi, DHO and OPRT cluster respectively with the corresponding proteobacterial counterparts. The phylogenetic analyses of DHOD and OMPDC also support the implications of the mosaic pyrimidine biosynthetic pathway in eukaryotes. The potential importance of the horizontal gene transfer(s) and endosymbiosis in establishing the mosaic pathway is discussed. << Less
Gene 257:209-222(2000) [PubMed] [EuropePMC]
This publication is cited by 1 other entry.
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Mechanism of flavin reduction in class 2 dihydroorotate dehydrogenases.
Fagan R.L., Nelson M.N., Pagano P.M., Palfey B.A.
Dihydroorotate dehydrogenases (DHODs) oxidize dihydroorotate (DHO) to orotate using the FMN prosthetic group to abstract a hydride equivalent from C6 and a protein residue (Ser for Class 2 DHODs) to deprotonate C5. The fundamental question of whether the scission of the two DHO C-H bonds is concer ... >> More
Dihydroorotate dehydrogenases (DHODs) oxidize dihydroorotate (DHO) to orotate using the FMN prosthetic group to abstract a hydride equivalent from C6 and a protein residue (Ser for Class 2 DHODs) to deprotonate C5. The fundamental question of whether the scission of the two DHO C-H bonds is concerted or stepwise was addressed for two Class 2 enzymes, those from Escherichia coli and Homo sapiens, by determining kinetic isotope effects on flavin reduction in anaerobic stopped-flow experiments. Isotope effects were determined for the E. coli enzyme at two pH values below a previously reported pKa controlling reduction [Palfey, B. A., Björnberg, O., and Jensen K. F. (2001) Biochemistry 40, 4381-4390] and were about 3-fold for DHO labeled at the 5-position, about 4-fold for DHO labeled at the 6-position, and about 6-7-fold for DHO labeled at both the 5- and 6-positions. These isotope effects are consistent with either a stepwise oxidation of DHO or a concerted mechanism with significant quantum mechanical tunneling. At a pH value above the pKa controlling reduction, no isotope effect was observed in E. coli DHOD for DHO deuterated at the 5-position (the proton donor in the reaction). This is consistent with a stepwise reaction; above the (kinetic) pKa, the deprotonation of C5 is fast enough that it does not contribute to the observed rate constant and, therefore, is not isotopically sensitive. All available information points to Ser acting as a component in a proton relay network which allows its transient deprotonation. The H. sapiens DHOD also appears to have a pKa near 9.4 controlling reduction, similar to that previously reported for the E. coli enzyme. Similar KIEs were obtained with the H. sapiens enzyme at a pH value below the pKa. << Less
Biochemistry 45:14926-14932(2006) [PubMed] [EuropePMC]
This publication is cited by 1 other entry.
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Purification and properties of the bovine liver mitochondrial dihydroorotate dehydrogenase.
Hines V., Keys L.D. III, Johnston M.
Dihydroorotate dehydrogenase has been purified 6,000-fold from bovine liver mitochondria to apparent homogeneity in six steps. Electrophoretic migration of the homogeneous enzyme on sodium dodecyl sulfate-polyacrylamide gels reveals a subunit Mr of 42,000. By contrast to the well-characterized, cy ... >> More
Dihydroorotate dehydrogenase has been purified 6,000-fold from bovine liver mitochondria to apparent homogeneity in six steps. Electrophoretic migration of the homogeneous enzyme on sodium dodecyl sulfate-polyacrylamide gels reveals a subunit Mr of 42,000. By contrast to the well-characterized, cytosolic dihydroorotate oxidases (EC 1.3.3.1), the purified bovine dehydrogenase is a dihydroorotate:ubiquinone oxidoreductase. Maximal rates of orotate formation are obtained using coenzymes Q6 or Q7 as cosubstrate electron acceptors. Concomitant with substrate oxidation, the enzyme will reduce simple quinones, such as benzoquinone, but at significantly lower rates (10-15%) than that obtained for reduction of coenzyme Q6. Enzyme-catalyzed substrate oxidation is not supported by molecular oxygen. The specificity of the purified enzyme for dihydropyrimidine substrates has also been explored. The methyl-, ethyl-, t-butyl-, and benzyl-S-dihydroorotates are substrates, but 1- and 3-methyl and 1,3-dimethyl methyl-S-dihydroorotates are not. Competitive inhibitors include product orotate, 5-methyl orotate, and racemic cis-5-methyl dihydroorotate. << Less
J. Biol. Chem. 261:11386-11392(1986) [PubMed] [EuropePMC]
This publication is cited by 1 other entry.
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Expression, purification, and characterization of histidine-tagged rat and human flavoenzyme dihydroorotate dehydrogenase.
Bader B., Knecht W., Fries M., Loffler M.
Mitochondrially bound dihydroorotate-ubiquinone oxidoreductase (dihydroorotate dehydrogenase, EC 1.3.99.11) catalyzes the fourth sequential step in the de novo synthesis of uridine monophosphate. Based on the recent functional expression of the complete rat dihydroorotate dehydrogenase by means of ... >> More
Mitochondrially bound dihydroorotate-ubiquinone oxidoreductase (dihydroorotate dehydrogenase, EC 1.3.99.11) catalyzes the fourth sequential step in the de novo synthesis of uridine monophosphate. Based on the recent functional expression of the complete rat dihydroorotate dehydrogenase by means of the baculovirus expression vector system in Trichoplusia ni cells, a procedure is described that allows the purification of baculovirus expressed enzyme protein fused to a carboxy-terminal tag of eight histidines. Extracts from mitochondria of Spodoptera frugiperda cells infected with the recombinant virus using Triton X-100 were loaded onto Ni2+-nitrilotriacetic acid agarose and histidine-tagged rat protein was selectively eluted with imidazole-containing buffer. In view of our previously published work, the quality of the electrophoretic homogenous rat enzyme was markedly improved; specific activity was 130-150 micromol dihydroorotate/min per milligram; and the stoichiometry of flavin content was 0.8-1.1 mol/mol protein. Efforts to generate mammalian dihydroorotate dehydrogenases with low production costs from bacteria resulted in successful overexpression of the carboxy-terminal-modified rat and human dihydroorotate dehydrogenase in XL-1 Blue cells. By employing the metal chelate affinity chromatography under native conditions, the histidine-tagged human enzyme was purified with a specific activity of 150 micromol/min/mg and the rat enzyme with 83 micromol/min/mg, respectively, at pH 8.0-8.1 optimum. Kinetic constants of the recombinant histidine-tagged rat enzyme from bacteria (dihydroorotate, Km = 14.6 micromol electron acceptor decylubiquinone, Km = 9.5 micromol) were close to those reported for the enzyme from insect cells, with or without the affinity tag. HPLC analyses identified flavin mononucleotide as cofactor of the rat enzyme; UV-vis and fluorometric analyses verified a flavin/protein ratio of 0.8-1.1 mol/mol. By spectral analyses of the functional flavin with the native human enzyme, the interaction of the pharmacological inhibitors Leflunomide and Brequinar with their target could be clarified as interference with the transfer of electrons from the flavin to the quinone. The combination of the bacterial expression system and metal chelate affinity chomatography offers an improved means to purify large quantities of mammalian membrane-bound dihydroorotate dehydrogenases which, by several criteria, possesses the same functional activities as non-histidine-tagged recombinant enzymes. << Less
Protein Expr Purif 13:414-422(1998) [PubMed] [EuropePMC]
This publication is cited by 1 other entry.
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The activity of Escherichia coli dihydroorotate dehydrogenase is dependent on a conserved loop identified by sequence homology, mutagenesis, and limited proteolysis.
Bjoernberg O., Gruener A.-C., Roepstorff P., Jensen K.F.
Dihydroorotate dehydrogenase catalyzes the oxidation of dihydroorotate to orotate. The enzyme from Escherichia coli was overproduced and characterized in comparison with the dimeric Lactococcus lactis A enzyme, whose structure is known. The two enzymes represent two distinct evolutionary families ... >> More
Dihydroorotate dehydrogenase catalyzes the oxidation of dihydroorotate to orotate. The enzyme from Escherichia coli was overproduced and characterized in comparison with the dimeric Lactococcus lactis A enzyme, whose structure is known. The two enzymes represent two distinct evolutionary families of dihydroorotate dehydrogenases, but sedimentation in sucrose gradients suggests a dimeric structure also of the E. coli enzyme. Product inhibition showed that the E. coli enzyme, in contrast to the L. lactis enzyme, has separate binding sites for dihydroorotate and the electron acceptor. Trypsin readily cleaved the E. coli enzyme into two fragments of 182 and 154 residues, respectively. Cleavage reduced the activity more than 100-fold but left other molecular properties, including the heat stability, intact. The trypsin cleavage site, at R182, is positioned in a conserved region that, in the L. lactis enzyme, forms a loop where a cysteine residue is very critical for activity. In the corresponding position, the enzyme from E. coli has a serine residue. Mutagenesis of this residue (S175) to alanine or cysteine reduced the activities 10000- and 500-fold, respectively. The S175C mutant was also defective with respect to substrate and product binding. Structural and mechanistic differences between the two different families of dihydroorotate dehydrogenase are discussed. << Less
Biochemistry 38:2899-2908(1999) [PubMed] [EuropePMC]
This publication is cited by 1 other entry.