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Literature summary extracted from

  • Bjoernberg, O.; Gruener, A.C.; Roepstorff, P.; Jensen, K.F.
    The activity of Escherichia coli dihydroorotate dehydrogenase is dependent on a conserved loop identified by sequence homology, mutagenesis, and limited proteolysis (1999), Biochemistry, 38, 2899-2908.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.3.5.2 overexpression of Escherichia coli dihyroorotate dehyrogenase in same strain, partially deleted for the chromosomal pyrD gene, clone selection followed by ampicillin and by complementation of the pyrimidine requirement Escherichia coli

Protein Variants

EC Number Protein Variants Comment Organism
1.3.5.2 C130A loss of activity Lactococcus lactis
1.3.5.2 C130S loss of activity Lactococcus lactis
1.3.5.2 S175A
-
 Lactococcus lactis
1.3.5.2 S175A very little activity Escherichia coli
1.3.5.2 S175C
-
 Lactococcus lactis
1.3.5.2 S175C sufficient activity, catalysis and binding of dihydrooratate are affected Escherichia coli

Inhibitors

EC Number Inhibitors Comment Organism Structure
1.3.5.2 Orotate competitive to dihydroorotate Escherichia coli

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.3.5.2 0.0146
-
 2,6-dichlorophenolindophenol S175C mutant Escherichia coli
1.3.5.2 0.0288
-
 S-dihydroorotate ubiquinone0 and 2,6-dichlorophenolindophenol mix Escherichia coli
1.3.5.2 0.03
-
 S-dihydroorotate 2,6-dichlorophenolindophenol sole electron acceptor Escherichia coli
1.3.5.2 0.04
-
 ubiquinone-0
-
 Escherichia coli
1.3.5.2 0.075
-
 ubiquinone-0
-
 Lactococcus lactis
1.3.5.2 0.0929
-
 S-dihydroorotate S175C mutant Escherichia coli

Localization

EC Number Localization Comment Organism GeneOntology No. Textmining
1.3.5.2 cytoplasm
-
 Escherichia coli 5737
-
1.3.5.2 membrane
-
 Escherichia coli 16020
-

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
1.3.5.2 36800
-
 2 * 37000, SDS-PAGE, DNA-sequence, 2 * 36800, MALDI MS Escherichia coli
1.3.5.2 37000
-
 2 * 37000, SDS-PAGE, DNA-sequence, 2 * 36800, MALDI MS Escherichia coli
1.3.5.2 37000
-
 2 * 37000, residue S175 is critical for activity Escherichia coli
1.3.5.2 67000
-
 gel filtration Escherichia coli
1.3.5.2 72000
-
 sedimentation analysis in sucrose density gradient in presence of detergent Escherichia coli
1.3.5.2 75000
-
 sedimentation in sucrose density gradients Escherichia coli

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.3.5.2 (S)-dihydroorotate + acceptor Escherichia coli fourth step in synthesis of pyrimidine nucleotides orotate + reduced acceptor
-
 ?
1.3.5.2 (S)-dihydroorotate + acceptor Lactococcus lactis fourth step in synthesis of pyrimidine nucleotides orotate + reduced acceptor
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.3.5.2 Escherichia coli
-
-
-
1.3.5.2 Escherichia coli
-
 family 2 enzyme
-
1.3.5.2 Lactococcus lactis
-
 only enzyme A
-

Oxidation Stability

EC Number Oxidation Stability Organism
1.3.5.2 reoxidation by O2 results in H2O2 which appears in the active site and is very harmful to a cysteine residue, H2O2 inactivates the enzyme Lactococcus lactis

Purification (Commentary)

EC Number Purification (Comment) Organism
1.3.5.2 ion-exchange, ammonium sulfate precipitation, gel filtration Escherichia coli

Reaction

EC Number Reaction Comment Organism Reaction ID
1.3.5.2 (S)-dihydroorotate + a quinone = orotate + a quinol ping-pong mechanism Escherichia coli
a
1.3.5.2 (S)-dihydroorotate + a quinone = orotate + a quinol different binding sites for dihydroorotate and the electron acceptor, two-site ping-pong mechanism. Cleavage site at R182 is conserved between the two major families of dihydroorotate dehydrogenases, it is positioned in a loop, which is crucial for catalysis but irrelevant for protein stability Escherichia coli
a
1.3.5.2 (S)-dihydroorotate + a quinone = orotate + a quinol one-site ping-pong mechanism, residues 129-137 form a flexible loop, responsible for substrate binding Lactococcus lactis
a

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
1.3.5.2 1.1 23 with 0.5 mM fumarate Escherichia coli
1.3.5.2 1.1 23 with 0.5 mM fumarate Lactococcus lactis
1.3.5.2 1.1 7.7 with O2 Escherichia coli
1.3.5.2 1.1 7.7 with O2 Lactococcus lactis
1.3.5.2 7.4 58 with 0.02 mM decylubiquinone Escherichia coli
1.3.5.2 7.4 58 with 0.02 mM decylubiquinone Lactococcus lactis
1.3.5.2 9.1 14.5 with 0.02 mM menadione Escherichia coli
1.3.5.2 9.1 14.5 with 0.02 mM menadione Lactococcus lactis
1.3.5.2 20 24 with 0.02 mM ubiquinone0 Escherichia coli
1.3.5.2 20 24 with 0.02 mM ubiquinone0 Lactococcus lactis
1.3.5.2 22
-
 with 0.02 mM 2,6-dichlorophenolindophenol Escherichia coli
1.3.5.2 22
-
 with 0.02 mM 2,6-dichlorophenolindophenol Lactococcus lactis

Storage Stability

EC Number Storage Stability Organism
1.3.5.2 -20°C, 10 mg/ml, 50 mM sodium phosphate, pH 7.0, 0.1 mM EDTA, 50% glycerol Escherichia coli

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.3.5.2 (S)-dihydroorotate + acceptor with ubiquinone-0, 2,6-dichlorophenolindophenol, menadione, decylubiquinone, fumarate and O2 as electron acceptor Escherichia coli orotate + reduced acceptor
-
 ?
1.3.5.2 (S)-dihydroorotate + acceptor with ubiquinone-0, 2,6-dichlorophenolindophenol, menadione, decylubiquinone, fumarate and O2 as electron acceptor Lactococcus lactis orotate + reduced acceptor
-
 ?
1.3.5.2 (S)-dihydroorotate + acceptor fourth step in synthesis of pyrimidine nucleotides Escherichia coli orotate + reduced acceptor
-
 ?
1.3.5.2 (S)-dihydroorotate + acceptor fourth step in synthesis of pyrimidine nucleotides Lactococcus lactis orotate + reduced acceptor
-
 ?
1.3.5.2 (S)-dihydroorotate + ubiquinone-0
-
 Escherichia coli orotate + ubiquinol
-
 r
1.3.5.2 (S)-dihydroorotate + ubiquinone-0
-
 Lactococcus lactis orotate + ubiquinol
-
 r
1.3.5.2 L-dihydroorotate + 2,6-dichlorophenolindophenol
-
 Escherichia coli orotate + reduced 2,6-dichlorophenolindophenol
-
 ?

Subunits

EC Number Subunits Comment Organism
1.3.5.2 ?
-
 Lactococcus lactis
1.3.5.2 dimer
-
 Lactococcus lactis
1.3.5.2 dimer 2 * 37000, SDS-PAGE, DNA-sequence, 2 * 36800, MALDI MS Escherichia coli
1.3.5.2 dimer 2 * 37000, residue S175 is critical for activity Escherichia coli

Synonyms

EC Number Synonyms Comment Organism
1.3.5.2 DHOD
-
 Escherichia coli
1.3.5.2 DHOD
-
 Lactococcus lactis

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.3.5.2 25
-
 comparison assay Escherichia coli
1.3.5.2 25
-
 comparison assay Lactococcus lactis
1.3.5.2 37
-
 assay at Escherichia coli
1.3.5.2 37
-
 assay at Lactococcus lactis

Temperature Stability [°C]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
1.3.5.2 37
-
 unstable with O2 Lactococcus lactis
1.3.5.2 50
-
 Tm-value Lactococcus lactis
1.3.5.2 70
-
 Tm-value Escherichia coli

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.3.5.2 8
-
 assay at Escherichia coli

Cofactor

EC Number Cofactor Comment Organism Structure
1.3.5.2 FMN
-
 Escherichia coli
1.3.5.2 FMN
-
 Lactococcus lactis

Ki Value [mM]

EC Number Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
1.3.5.2 0.0134
-
 Orotate
-
 Escherichia coli