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Literature summary extracted from

  • Regev, O.; Korman, M.; Hecht, N.; Roth, Z.; Forer, N.; Zarivach, R.; Gur, E.
    An extended loop of the Pup ligase, PafA, mediates interaction with protein targets (2016), J. Mol. Biol., 428, 4143-4153 .
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
6.3.1.19 recombinant expression of His6-tagged enzyme in Escherichia coli strain ER2566 Corynebacterium glutamicum

Protein Variants

EC Number Protein Variants Comment Organism
6.3.1.19 A196S site-directed mutagenesis, the mutant shows increased pupylation activity with substrate PanB compared to wild-type Corynebacterium glutamicum
6.3.1.19 D208A site-directed mutagenesis, the mutant shows reduced pupylation activity with substrate PanB compared to wild-type Corynebacterium glutamicum
6.3.1.19 D64N site-directed mutagenesis, a catalytically inactive mutant Corynebacterium glutamicum
6.3.1.19 E209A site-directed mutagenesis, the mutant shows highly reduced pupylation activity with substrate PanB compared to wild-type Corynebacterium glutamicum
6.3.1.19 E70A site-directed mutagenesis, a catalytically inactive mutant Corynebacterium glutamicum
6.3.1.19 N205A site-directed mutagenesis, the mutant shows reduced pupylation activity with substrate PanB compared to wild-type Corynebacterium glutamicum
6.3.1.19 R199A site-directed mutagenesis, the mutant shows highly reduced pupylation activity with substrate PanB compared to wild-type Corynebacterium glutamicum
6.3.1.19 R201A site-directed mutagenesis, pupylation catalytically inactive mutant Corynebacterium glutamicum
6.3.1.19 R207A site-directed mutagenesis, the mutant shows highly reduced pupylation activity with substrate PanB compared to wild-type Corynebacterium glutamicum
6.3.1.19 R207A site-directed mutagenesis, whereas extensive protein pupylation occurs with the wild-type enzyme, the R207A mutant catalyzes protein pupylation much more slowly. PafA residue R207 stabilizes the interaction of the enzyme with PanB, the R207A mutant amidates PupE and the wild-type variant Corynebacterium glutamicum
6.3.1.19 T197A site-directed mutagenesis, the mutant shows reduced pupylation activity with substrate PanB compared to wild-type Corynebacterium glutamicum
6.3.1.19 T198A site-directed mutagenesis, the mutant shows highly reduced pupylation activity with substrate PanB compared to wild-type Corynebacterium glutamicum
6.3.1.19 V65S site-directed mutagenesis, the mutant shows reduced pupylation activity with substrate PanB compared to wild-type Corynebacterium glutamicum

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
6.3.1.19 additional information
-
 additional information Michaelis-Menten kinetic analysis of poly-pupylation, overview Corynebacterium glutamicum
6.3.1.19 0.084
-
 [PanB]-L-lysine pH 7.5, 30°C, recombinant His-tagged enzyme Corynebacterium glutamicum

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
6.3.1.19 Mg2+ required Corynebacterium glutamicum

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
6.3.1.19 ATP + [prokaryotic ubiquitin-like protein]-L-glutamate + [protein]-L-lysine Corynebacterium glutamicum
-
 ADP + phosphate + N6-([prokaryotic ubiquitin-like protein]-gamma-L-glutamyl)-[protein]-L-lysine
-
 ?
6.3.1.19 ATP + [prokaryotic ubiquitin-like protein]-L-glutamate + [protein]-L-lysine Corynebacterium glutamicum ATCC 13032 / DSM 20300 / JCM 1318 / LMG 3730 / NCIMB 10025
-
 ADP + phosphate + N6-([prokaryotic ubiquitin-like protein]-gamma-L-glutamyl)-[protein]-L-lysine
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
6.3.1.19 Corynebacterium glutamicum Q8NQE1
-
-
6.3.1.19 Corynebacterium glutamicum ATCC 13032 / DSM 20300 / JCM 1318 / LMG 3730 / NCIMB 10025 Q8NQE1
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
6.3.1.19 recombinant His6-tagged enzyme from Escherichia coli strain ER2566 by nickel affinity chormatography Corynebacterium glutamicum

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
6.3.1.19 ATP + [prokaryotic ubiquitin-like protein]-L-glutamate + [PanB]-L-lysine Mycobacterium tuberculosis PanB is a model substrate of PafA, in silico docking analysis reveals interaction via PafA residue arginine 207 Corynebacterium glutamicum ADP + phosphate + N6-([prokaryotic ubiquitin-like protein]-gamma-L-glutamyl)-[PanB]-L-lysine
-
 ?
6.3.1.19 ATP + [prokaryotic ubiquitin-like protein]-L-glutamate + [PanB]-L-lysine Mycobacterium tuberculosis PanB is a model substrate of PafA, in silico docking analysis reveals interaction via PafA residue arginine 207 Corynebacterium glutamicum ATCC 13032 / DSM 20300 / JCM 1318 / LMG 3730 / NCIMB 10025 ADP + phosphate + N6-([prokaryotic ubiquitin-like protein]-gamma-L-glutamyl)-[PanB]-L-lysine
-
 ?
6.3.1.19 ATP + [prokaryotic ubiquitin-like protein]-L-glutamate + [protein]-L-lysine
-
 Corynebacterium glutamicum ADP + phosphate + N6-([prokaryotic ubiquitin-like protein]-gamma-L-glutamyl)-[protein]-L-lysine
-
 ?
6.3.1.19 ATP + [prokaryotic ubiquitin-like protein]-L-glutamate + [protein]-L-lysine the Mycobacterium smegmatis Pup variant used presents a glutamate at its C-terminus (PupE) and, as such, can be readily conjugated to target proteins by PafA Corynebacterium glutamicum ADP + phosphate + N6-([prokaryotic ubiquitin-like protein]-gamma-L-glutamyl)-[protein]-L-lysine
-
 ?
6.3.1.19 ATP + [prokaryotic ubiquitin-like protein]-L-glutamate + [protein]-L-lysine
-
 Corynebacterium glutamicum ATCC 13032 / DSM 20300 / JCM 1318 / LMG 3730 / NCIMB 10025 ADP + phosphate + N6-([prokaryotic ubiquitin-like protein]-gamma-L-glutamyl)-[protein]-L-lysine
-
 ?
6.3.1.19 ATP + [prokaryotic ubiquitin-like protein]-L-glutamate + [protein]-L-lysine the Mycobacterium smegmatis Pup variant used presents a glutamate at its C-terminus (PupE) and, as such, can be readily conjugated to target proteins by PafA Corynebacterium glutamicum ATCC 13032 / DSM 20300 / JCM 1318 / LMG 3730 / NCIMB 10025 ADP + phosphate + N6-([prokaryotic ubiquitin-like protein]-gamma-L-glutamyl)-[protein]-L-lysine
-
 ?
6.3.1.19 additional information PafA substrate docking study, overview. PafA can use ammoniumions as targets for pupylation. The reaction catalyzed is in fact the conversion of PupE C-terminal glutamate into a glutamine, that is, PupE amidation. The docking of a small molecule like ammonium in the PafA active site cannot be supported by auxiliary interactions. PafA residue R207 stabilizes the interaction of the enzyme with PanB, the R207A mutant amidates PupE and the wild-type variant Corynebacterium glutamicum ?
-
 ?
6.3.1.19 additional information PafA substrate docking study, overview. PafA can use ammoniumions as targets for pupylation. The reaction catalyzed is in fact the conversion of PupE C-terminal glutamate into a glutamine, that is, PupE amidation. The docking of a small molecule like ammonium in the PafA active site cannot be supported by auxiliary interactions. PafA residue R207 stabilizes the interaction of the enzyme with PanB, the R207A mutant amidates PupE and the wild-type variant Corynebacterium glutamicum ATCC 13032 / DSM 20300 / JCM 1318 / LMG 3730 / NCIMB 10025 ?
-
 ?

Synonyms

EC Number Synonyms Comment Organism
6.3.1.19 PafA
-
 Corynebacterium glutamicum
6.3.1.19 Pup ligase
-
 Corynebacterium glutamicum

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
6.3.1.19 30
-
 assay at Corynebacterium glutamicum

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
6.3.1.19 7.5
-
 assay at Corynebacterium glutamicum

Cofactor

EC Number Cofactor Comment Organism Structure
6.3.1.19 ATP
-
 Corynebacterium glutamicum

General Information

EC Number General Information Comment Organism
6.3.1.19 physiological function pupylation, the bacterial equivalent of ubiquitylation, involves the conjugation of a prokaryotic ubiquitin-like protein (Pup) to protein targets. In contrast to the ubiquitin system, where many ubiquitin ligases exist, a single bacterial ligase, PafA, catalyzes the conjugation of Pup to a wide array of protein targets Corynebacterium glutamicum