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Literature summary for 1.3.5.2 extracted from
- 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.
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| 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
| Protein Variants | Comment | Organism |
|---|---|---|
| C130A | loss of activity | Lactococcus lactis |
| C130S | loss of activity | Lactococcus lactis |
| S175A | - |
Lactococcus lactis |
| S175A | very little activity | Escherichia coli |
| S175C | - |
Lactococcus lactis |
| S175C | sufficient activity, catalysis and binding of dihydrooratate are affected | Escherichia coli |
Inhibitors
| Inhibitors | Comment | Organism | Structure |
|---|---|---|---|
| Orotate | competitive to dihydroorotate | Escherichia coli |  |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.0146 | - |
2,6-dichlorophenolindophenol | S175C mutant | Escherichia coli | |
| 0.0288 | - |
S-dihydroorotate | ubiquinone0 and 2,6-dichlorophenolindophenol mix | Escherichia coli | |
| 0.03 | - |
S-dihydroorotate | 2,6-dichlorophenolindophenol sole electron acceptor | Escherichia coli | |
| 0.04 | - |
ubiquinone-0 | - |
Escherichia coli | |
| 0.075 | - |
ubiquinone-0 | - |
Lactococcus lactis | |
| 0.0929 | - |
S-dihydroorotate | S175C mutant | Escherichia coli | |
Localization
| Localization | Comment | Organism | GeneOntology No. | Textmining |
|---|---|---|---|---|
| cytoplasm | - |
Escherichia coli | 5737 | - |
| membrane | - |
Escherichia coli | 16020 | - |
Molecular Weight [Da]
| Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
|---|---|---|---|
| 36800 | - |
2 * 37000, SDS-PAGE, DNA-sequence, 2 * 36800, MALDI MS | Escherichia coli |
| 37000 | - |
2 * 37000, SDS-PAGE, DNA-sequence, 2 * 36800, MALDI MS | Escherichia coli |
| 37000 | - |
2 * 37000, residue S175 is critical for activity | Escherichia coli |
| 67000 | - |
gel filtration | Escherichia coli |
| 72000 | - |
sedimentation analysis in sucrose density gradient in presence of detergent | Escherichia coli |
| 75000 | - |
sedimentation in sucrose density gradients | Escherichia coli |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| (S)-dihydroorotate + acceptor | Escherichia coli | fourth step in synthesis of pyrimidine nucleotides | orotate + reduced acceptor | - |
? | |
| (S)-dihydroorotate + acceptor | Lactococcus lactis | fourth step in synthesis of pyrimidine nucleotides | orotate + reduced acceptor | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Escherichia coli | - |
- |
- |
| Escherichia coli | - |
family 2 enzyme | - |
| Lactococcus lactis | - |
only enzyme A | - |
Oxidation Stability
| Oxidation Stability | Organism |
|---|---|
| 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)
| Purification (Comment) | Organism |
|---|---|
| ion-exchange, ammonium sulfate precipitation, gel filtration | Escherichia coli |
Reaction
| Reaction | Comment | Organism | Reaction ID |
|---|---|---|---|
| (S)-dihydroorotate + a quinone = orotate + a quinol | ping-pong mechanism | Escherichia coli | |
| (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 | |
| (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 |
Specific Activity [micromol/min/mg]
| Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
|---|---|---|---|
| 1.1 | 23 | with 0.5 mM fumarate | Escherichia coli |
| 1.1 | 23 | with 0.5 mM fumarate | Lactococcus lactis |
| 1.1 | 7.7 | with O2 | Escherichia coli |
| 1.1 | 7.7 | with O2 | Lactococcus lactis |
| 7.4 | 58 | with 0.02 mM decylubiquinone | Escherichia coli |
| 7.4 | 58 | with 0.02 mM decylubiquinone | Lactococcus lactis |
| 9.1 | 14.5 | with 0.02 mM menadione | Escherichia coli |
| 9.1 | 14.5 | with 0.02 mM menadione | Lactococcus lactis |
| 20 | 24 | with 0.02 mM ubiquinone0 | Escherichia coli |
| 20 | 24 | with 0.02 mM ubiquinone0 | Lactococcus lactis |
| 22 | - |
with 0.02 mM 2,6-dichlorophenolindophenol | Escherichia coli |
| 22 | - |
with 0.02 mM 2,6-dichlorophenolindophenol | Lactococcus lactis |
Storage Stability
| Storage Stability | Organism |
|---|---|
| -20°C, 10 mg/ml, 50 mM sodium phosphate, pH 7.0, 0.1 mM EDTA, 50% glycerol | Escherichia coli |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| (S)-dihydroorotate + acceptor | with ubiquinone-0, 2,6-dichlorophenolindophenol, menadione, decylubiquinone, fumarate and O2 as electron acceptor | Escherichia coli | orotate + reduced acceptor | - |
? | |
| (S)-dihydroorotate + acceptor | with ubiquinone-0, 2,6-dichlorophenolindophenol, menadione, decylubiquinone, fumarate and O2 as electron acceptor | Lactococcus lactis | orotate + reduced acceptor | - |
? | |
| (S)-dihydroorotate + acceptor | fourth step in synthesis of pyrimidine nucleotides | Escherichia coli | orotate + reduced acceptor | - |
? | |
| (S)-dihydroorotate + acceptor | fourth step in synthesis of pyrimidine nucleotides | Lactococcus lactis | orotate + reduced acceptor | - |
? | |
| (S)-dihydroorotate + ubiquinone-0 | - |
Escherichia coli | orotate + ubiquinol | - |
r | |
| (S)-dihydroorotate + ubiquinone-0 | - |
Lactococcus lactis | orotate + ubiquinol | - |
r | |
| L-dihydroorotate + 2,6-dichlorophenolindophenol | - |
Escherichia coli | orotate + reduced 2,6-dichlorophenolindophenol | - |
? | |
Subunits
| Subunits | Comment | Organism |
|---|---|---|
| ? | - |
Lactococcus lactis |
| dimer | - |
Lactococcus lactis |
| dimer | 2 * 37000, SDS-PAGE, DNA-sequence, 2 * 36800, MALDI MS | Escherichia coli |
| dimer | 2 * 37000, residue S175 is critical for activity | Escherichia coli |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| DHOD | - |
Escherichia coli |
| DHOD | - |
Lactococcus lactis |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 25 | - |
comparison assay | Escherichia coli |
| 25 | - |
comparison assay | Lactococcus lactis |
| 37 | - |
assay at | Escherichia coli |
| 37 | - |
assay at | Lactococcus lactis |
Temperature Stability [°C]
| Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 37 | - |
unstable with O2 | Lactococcus lactis |
| 50 | - |
Tm-value | Lactococcus lactis |
| 70 | - |
Tm-value | Escherichia coli |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 8 | - |
assay at | Escherichia coli |
Cofactor
| Cofactor | Comment | Organism | Structure |
|---|---|---|---|
| FMN | - |
Escherichia coli | |
| FMN | - |
Lactococcus lactis | |
Ki Value [mM]
| Ki Value [mM] | Ki Value maximum [mM] | Inhibitor | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.0134 | - |
Orotate | - |
Escherichia coli | |
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