Application | Comment | Organism |
---|---|---|
drug development | the Pup-proteasome enzymes are essential for full virulence and persistence in the mammalian host. As such, the Pup-proteasome enzymes are potential targets for development of antituberculosis therapeutics. Such development requires sensitive and robust assays for measurements of enzymatic activities and the effect of examined inhibitors. An in vitro activity assay for Dop, the first enzyme in the Pup-proteasome system is described, that is ased on fluorescence anisotropy measurements. This assay is simple, sensitive, and compatible with a high-throughput format for screening purposes and can be reliably and conveniently used for detailed kinetic measurements of Dop activity | Mycobacterium tuberculosis |
Cloned (Comment) | Organism |
---|---|
expressed in Escherichia coli ER2566 from plasmid pET11a | Mycolicibacterium smegmatis |
expressed in Escherichia coli ER2566 from plasmid pET11a | Acidothermus cellulolyticus |
Protein Variants | Comment | Organism |
---|---|---|
D95A | inactive mutant enzyme | Mycolicibacterium smegmatis |
D95A | inactive mutant enzyme | Acidothermus cellulolyticus |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.001 | - |
[prokaryotic ubiquitin-like protein]-fluorescein-5-carboxamide-L-lysine | pH 8.0, 23°C | Mycolicibacterium smegmatis |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Acidothermus cellulolyticus | A0LU48 | - |
- |
Acidothermus cellulolyticus 11B | A0LU48 | - |
- |
Acidothermus cellulolyticus ATCC 43068 | A0LU48 | - |
- |
Mycobacterium tuberculosis | - |
- |
- |
Mycolicibacterium smegmatis | - |
- |
- |
Purification (Comment) | Organism |
---|---|
- |
Mycolicibacterium smegmatis |
- |
Acidothermus cellulolyticus |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
[prokaryotic ubiquitin-like protein]-fluorescein-5-carboxamide-L-lysine + H2O | hydrolysis of the isopeptide bond in Pup-Fl and release the fluorescein-5-carboxamide lysine moiety | Mycolicibacterium smegmatis | [prokaryotic ubiquitin-like protein]-L-lysine + 5-carboxyfluorescein | - |
? | |
[prokaryotic ubiquitin-like protein]-fluorescein-5-carboxamide-L-lysine + H2O | hydrolysis of the isopeptide bond in Pup-Fl and release the fluorescein-5-carboxamide lysine moiety | Acidothermus cellulolyticus | [prokaryotic ubiquitin-like protein]-L-lysine + 5-carboxyfluorescein | - |
? | |
[prokaryotic ubiquitin-like protein]-fluorescein-5-carboxamide-L-lysine + H2O | hydrolysis of the isopeptide bond in Pup-Fl and release the fluorescein-5-carboxamide lysine moiety | Acidothermus cellulolyticus 11B | [prokaryotic ubiquitin-like protein]-L-lysine + 5-carboxyfluorescein | - |
? | |
[prokaryotic ubiquitin-like protein]-fluorescein-5-carboxamide-L-lysine + H2O | hydrolysis of the isopeptide bond in Pup-Fl and release the fluorescein-5-carboxamide lysine moiety | Acidothermus cellulolyticus ATCC 43068 | [prokaryotic ubiquitin-like protein]-L-lysine + 5-carboxyfluorescein | - |
? |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
ATP | - |
Mycobacterium tuberculosis | |
ATP | - |
Acidothermus cellulolyticus | |
ATP | Kapp: 0.0089 mM | Mycolicibacterium smegmatis |
General Information | Comment | Organism |
---|---|---|
physiological function | the Pup-proteasome enzymes are essential for full virulence and persistence in the mammalian host | Mycobacterium tuberculosis |