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Literature summary for 3.5.1.119 extracted from

  • Hecht, N.; Gur, E.
    Development of a fluorescence anisotropy-based assay for Dop, the first enzyme in the pupylation pathway (2015), Anal. Biochem., 485, 97-101.
    View publication on PubMed
Search for more literature for 3.5.1.119

Application

Application Comment Organism
analysis development of an in vitro activity assay for Dop, based on fluorescence anisotropy measurements. The assay is simple, sensitive, and compatible with a high-throughput format for screening purposes. It can be reliably and conveniently used for detailed kinetic measurements of Dop activity and can be employed with other Dop enzymes Mycolicibacterium smegmatis
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(Commentary)

Cloned (Comment) Organism
-
 Mycolicibacterium smegmatis
expressed in Escherichia coli ER2566 from plasmid pET11a Acidothermus cellulolyticus
expressed in Escherichia coli ER2566 from plasmid pET11a Mycolicibacterium smegmatis

Protein Variants

Protein Variants Comment Organism
D95A inactive mutant enzyme Mycolicibacterium smegmatis
D95A inactive mutant enzyme Acidothermus cellulolyticus

KM Value [mM]

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

Organism UniProt Comment Textmining
Acidothermus cellulolyticus A0LU48
-
-
Acidothermus cellulolyticus 11B A0LU48
-
-
Acidothermus cellulolyticus ATCC 43068 A0LU48
-
-
Mycobacterium tuberculosis
-
-
-
Mycolicibacterium smegmatis
-
-
-
Mycolicibacterium smegmatis A0QZ49
-
-
Mycolicibacterium smegmatis ATCC 700084 A0QZ49
-
-

Purification (Commentary)

Purification (Comment) Organism
-
 Acidothermus cellulolyticus
-
 Mycolicibacterium smegmatis

Substrates and Products (Substrate)

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

Cofactor Comment Organism Structure
ATP
-
 Mycobacterium tuberculosis
ATP Kapp: 0.0089 mM Mycolicibacterium smegmatis
ATP
-
 Acidothermus cellulolyticus

General Information

General Information Comment Organism
physiological function the Pup-proteasome enzymes are essential for full virulence and persistence in the mammalian host Mycobacterium tuberculosis