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Literature summary for 1.3.1.14 extracted from
- Biochemical characterization of the heteromeric Bacillus subtilis dihydroorotate dehydrogenase and its isolated subunits (1999), Arch. Biochem. Biophys., 371, 191-201.
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
- |
Bacillus subtilis |
| expression in Escherichia coliBL21 DE3, expression of pyrDI alone and coexpression with pyrDII | Bacillus subtilis |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| C220A | pyrDII mutant, conserved, essential for activity | Bacillus subtilis |
| C225A | pyrDII mutant conserved, essential for activity | Bacillus subtilis |
| C228A | pyrDII mutant conserved, essential for activity | Bacillus subtilis |
| C230A | pyrDII mutant nonconserved | Bacillus subtilis |
| C243A | pyrDII mutant conserved, essential for activity | Bacillus subtilis |
Inhibitors
| Inhibitors | Comment | Organism | Structure |
|---|---|---|---|
| Orotate | - |
Bacillus subtilis |  |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.027 | - |
S-dihydroorotate | holoenzyme with 2,6-dichlorophenolindophenol | Bacillus subtilis | |
| 0.0317 | - |
S-dihydroorotate | holoenzyme with menadione | Bacillus subtilis | |
| 0.5 | 1 | S-dihydroorotate | PyrDI | Bacillus subtilis | |
Molecular Weight [Da]
| Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
|---|---|---|---|
| 28000 | - |
2 * 33000 + 2 * 28000 | Bacillus subtilis |
| 28099 | - |
heterotetramer 2 * 33094 and 2 * 28099 predicted from seqeuence, 2 * 38000 and 2 * 31000, SDS-PAGE | Bacillus subtilis |
| 31000 | - |
heterotetramer 2 * 33094 and 2 * 28099 predicted from seqeuence, 2 * 38000 and 2 * 31000, SDS-PAGE | Bacillus subtilis |
| 33000 | - |
2 * 33000 + 2 * 28000 | Bacillus subtilis |
| 33094 | - |
heterotetramer 2 * 33094 and 2 * 28099 predicted from seqeuence, 2 * 38000 and 2 * 31000, SDS-PAGE | Bacillus subtilis |
| 38000 | - |
heterotetramer 2 * 33094 and 2 * 28099 predicted from seqeuence, 2 * 38000 and 2 * 31000, SDS-PAGE | Bacillus subtilis |
| 85000 | - |
native molecular mass of PyrDI | Bacillus subtilis |
| 114000 | - |
gel filtration | Bacillus subtilis |
| 114000 | - |
molecular mass of the holoenzyme, calculated mass 122400 Da | Bacillus subtilis |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| (S)-dihydroorotate + acceptor | Bacillus subtilis | fourth step in UMP-biosynthesis | orotate + reduced acceptor | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Bacillus subtilis | - |
- |
- |
| Mus musculus | - |
Ehrlich ascites tumor cells | - |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| anion-exchange, gel filtration | Bacillus subtilis |
Reaction
| Reaction | Comment | Organism | Reaction ID |
|---|---|---|---|
| (S)-dihydroorotate + NAD+ = orotate + NADH + H+ | PyrDI contains the dihydroorotate-binding site, but PyrDII is required for full activity in vivo. Holoenzyme joins a NAD+-reductase activity | Bacillus subtilis |
Renatured (Commentary)
| Renatured (Comment) | Organism |
|---|---|
| activity can be recovered by mixing the purified subunits | Bacillus subtilis |
| PyrDII-containing inclusion bodies are denatured and refolded through dialysis into buffer | Bacillus subtilis |
Storage Stability
| Storage Stability | Organism |
|---|---|
| 4°C, holoenzyme, stable for many days | Bacillus subtilis |
| on ice at room temperature, PyrDI, gradually loses activity over a period of hours | Bacillus subtilis |
| room temperature, holoenzyme, stable for many hours | Bacillus subtilis |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| (S)-dihydroorotate + acceptor | different specific activities with potassium ferricyanide, O2, fumarate and NAD+ as electron acceptors for PyrDI and the holoenzyme | Bacillus subtilis | orotate + reduced acceptor | - |
? | |
| (S)-dihydroorotate + acceptor | fourth step in UMP-biosynthesis | Bacillus subtilis | orotate + reduced acceptor | - |
? | |
| dihydroorotate + acceptor | acceptor: NAD+ | Bacillus subtilis | orotate + reduced acceptor | - |
? | |
| dihydroorotate + acceptor | acceptor: menadione | Bacillus subtilis | orotate + reduced acceptor | - |
? | |
| dihydroorotate + acceptor | activity measurement in permeabilized Ehrlich ascites tumor cells, acceptor: nitroblue tetrazolium | Mus musculus | orotate + reduced acceptor | - |
? | |
Subunits
| Subunits | Comment | Organism |
|---|---|---|
| tetramer | heterotetramer 2 * 33094 and 2 * 28099 predicted from seqeuence, 2 * 38000 and 2 * 31000, SDS-PAGE | Bacillus subtilis |
| tetramer | 2 * 33000 + 2 * 28000 | Bacillus subtilis |
Cofactor
| Cofactor | Comment | Organism | Structure |
|---|---|---|---|
| FAD | 1 mol PyrDII binds 1 mol FAD and 1 mol [2Fe-2S] | Bacillus subtilis | |
| FAD | 1 mol/mol of subunit PyrDII | Bacillus subtilis | |
| FMN | PyrDI is an FMN-containing iron-sulfur flavoprotein, 1 FMN molecule per PyrDI molecule | Bacillus subtilis | |
| NAD+ | for holoenzyme | Bacillus subtilis | |
Ki Value [mM]
| Ki Value [mM] | Ki Value maximum [mM] | Inhibitor | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.0206 | - |
Orotate | holoenzyme | Bacillus subtilis | |
| 1 | - |
Orotate | PyrDI | Bacillus subtilis | |
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