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Literature summary for 3.5.2.3 extracted from
- High-level expression, purification, and characterization of Staphylococcus aureus dihydroorotase (PyrC) as a cleavable His-SUMO fusion (2013), Protein Expr. Purif., 88, 98-106.
Application
| Application | Comment | Organism |
|---|---|---|
| drug development | the essential enzyme is a target for antibacterial drug design | Staphylococcus aureus |
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| 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 | Staphylococcus aureus |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| additional information | - |
additional information | forward and reverse kinetic rate constants for both tagged and wild-type enzyme, kinetic analysis, overview | Staphylococcus aureus | |
| 0.118 | - |
(S)-dihydroorotate | pH 8.3, 25°C, recombinant detagged enzyme | Staphylococcus aureus |  |
| 0.167 | - |
(S)-dihydroorotate | pH 8.3, 25°C, recombinant tagged enzyme | Staphylococcus aureus | |
| 0.334 | - |
N-carbamoyl-L-aspartate | pH 8.3, 25°C, recombinant tagged enzyme | Staphylococcus aureus | |
| 0.348 | - |
N-carbamoyl-L-aspartate | pH 8.3, 25°C, recombinant detagged enzyme | Staphylococcus aureus | |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| Zn2+ | dihydroorotase enzyme is a di-metalloenzyme that is dependent upon two zinc atoms in the active site for activity | Staphylococcus aureus | |
Molecular Weight [Da]
| Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
|---|---|---|---|
| 46370 | - |
2 * 46370, sequence calculation | Staphylococcus aureus |
| 46500 | - |
detagged recombinant enzyme, gel filtration | Staphylococcus aureus |
| 60700 | - |
His-SUMO-tagged recombinant enzyme, gel filtration | Staphylococcus aureus |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| (S)-dihydroorotate + H2O | Staphylococcus aureus | - |
N-carbamoyl-L-aspartate | - |
r | |
| (S)-dihydroorotate + H2O | Staphylococcus aureus MW2 | - |
N-carbamoyl-L-aspartate | - |
r | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Staphylococcus aureus | P65907 | methicillin-resistant strain, gene pyrC | - |
| Staphylococcus aureus MW2 | P65907 | methicillin-resistant strain, gene pyrC | - |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| 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 | Staphylococcus aureus |
Specific Activity [micromol/min/mg]
| Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
|---|---|---|---|
| 0.68 | - |
purified recombinant detagged enzyme, pH 8.3, 25°C | Staphylococcus aureus |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| (S)-dihydroorotate + H2O | - |
Staphylococcus aureus | N-carbamoyl-L-aspartate | - |
r | |
| (S)-dihydroorotate + H2O | - |
Staphylococcus aureus MW2 | N-carbamoyl-L-aspartate | - |
r | |
Subunits
| Subunits | Comment | Organism |
|---|---|---|
| homodimer | 2 * 46370, sequence calculation | Staphylococcus aureus |
| More | the His-SUMO tag does not interfere with SaPyrC dimerization | Staphylococcus aureus |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| pyrC | - |
Staphylococcus aureus |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 25 | - |
assay at | Staphylococcus aureus |
Turnover Number [1/s]
| Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 1.55 | - |
(S)-dihydroorotate | pH 8.3, 25°C, recombinant detagged enzyme | Staphylococcus aureus | |
| 1.87 | - |
(S)-dihydroorotate | pH 8.3, 25°C, recombinant tagged enzyme | Staphylococcus aureus | |
| 1.9 | - |
N-carbamoyl-L-aspartate | pH 8.3, 25°C, recombinant tagged enzyme | Staphylococcus aureus | |
| 2.48 | - |
N-carbamoyl-L-aspartate | pH 8.3, 25°C, recombinant detagged enzyme | Staphylococcus aureus | |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 6 | - |
cyclization reaction | Staphylococcus aureus |
| 9 | - |
hydrolysis reaction | Staphylococcus aureus |
General Information
| General Information | Comment | Organism |
|---|---|---|
| metabolism | third enzyme in the bacterial de novo pyrimidine biosynthesis pathway, overview | Staphylococcus aureus |
kcat/KM [mM/s]
| kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 5.69 | - |
N-carbamoyl-L-aspartate | pH 8.3, 25°C, recombinant tagged enzyme | Staphylococcus aureus | |
| 7.13 | - |
N-carbamoyl-L-aspartate | pH 8.3, 25°C, recombinant detagged enzyme | Staphylococcus aureus | |
| 11.2 | - |
(S)-dihydroorotate | pH 8.3, 25°C, recombinant tagged enzyme | Staphylococcus aureus | |
| 13.1 | - |
(S)-dihydroorotate | pH 8.3, 25°C, recombinant detagged enzyme | Staphylococcus aureus | |
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