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Literature summary for 6.3.1.19 extracted from

  • Chen, X.; Li, C.; Wang, L.; Liu, Y.; Li, C.; Zhang, J.
    The mechanism of Mycobacterium smegmatis PafA self-pupylation (2016), PLoS ONE, 11, e0151021 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
gene pafA, recombinant expression of N-terminal His6-tagged enzyme in Mycobacterium smegmatis, and recombinant expression of N-terminal His6-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3), recombinant expression of HA- or Strep-tagged enzyme mutants in Mycobacterium smegmatis Mycolicibacterium smegmatis

Protein Variants

Protein Variants Comment Organism
E9A site-directed mutagenesis, the PafA mutant without ligase activity cannot be pupylated Mycolicibacterium smegmatis
K31R site-directed mutagenesis, the mutation attenuates the poly-pupylation of PafA Mycolicibacterium smegmatis
K320R site-directed mutagenesis, the self-pupylation of PafA K320R mutant is not totally eliminated, suggesting that PafA can be poorly modified by Pup on other lysine residue(s) besides K320 in the in vitro reaction system. Wild-type PafA is less stable than PafA K320R, which cannot be pupylated in vivo Mycolicibacterium smegmatis
K61R site-directed mutagenesis, the mutation attenuates the poly-pupylation of PafA Mycolicibacterium smegmatis
K7R site-directed mutagenesis, the mutation attenuates the poly-pupylation of PafA Mycolicibacterium smegmatis
additional information Mycobacterium smegmatis strains with the deletion of the 20S proteasome beta subunit or pafA gene are constructed by homologous recombination with p1NIL and pGOAL19 plasmids, respectively Mycolicibacterium smegmatis

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ required Mycolicibacterium smegmatis

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
ATP + [prokaryotic ubiquitin-like protein]-L-glutamate + [protein]-L-lysine Mycolicibacterium smegmatis
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ADP + phosphate + N6-([prokaryotic ubiquitin-like protein]-gamma-L-glutamyl)-[protein]-L-lysine
-
?
ATP + [prokaryotic ubiquitin-like protein]-L-glutamate + [protein]-L-lysine Mycolicibacterium smegmatis ATCC 700084 / mc2155
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ADP + phosphate + N6-([prokaryotic ubiquitin-like protein]-gamma-L-glutamyl)-[protein]-L-lysine
-
?

Organism

Organism UniProt Comment Textmining
Mycolicibacterium smegmatis A0QZ42
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Mycolicibacterium smegmatis ATCC 700084 / mc2155 A0QZ42
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-

Purification (Commentary)

Purification (Comment) Organism
recombinant N-terminal His6-tagged wild-type and mutant enzymes from Mycobacterium smegmatis or Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration Mycolicibacterium smegmatis

Reaction

Reaction Comment Organism Reaction ID
ATP + [prokaryotic ubiquitin-like protein]-L-glutamate + [protein]-L-lysine = ADP + phosphate + N6-([prokaryotic ubiquitin-like protein]-gamma-L-glutamyl)-[protein]-L-lysine PafA catalyzes a two-step reaction by forming a gamma-glutamyl phosphate-mixed anhydride intermediate on the C-terminal glutamate of PupE by hydrolyzing ATP, followed by attaching it to nucleophilic substrates by catalyzing the formation of isopeptide bonds between PupE C-terminal glutamate gamma-carboxylate and the side chain of protein substrate lysine residues, mechanism of PafA self-pupylation, overview. K320 is the major target residue for the pupylation of PafA. During the self-pupylation of PafA, the attachment of the first Pup to PafA is catalyzed by the other PafA molecule through an intermolecular reaction, while the formation of the polymeric Pup chain is carried out in an intramolecular manner through the internal ligase activity of the already pupylated PafA. Among the three lysine residues, K7, K31 and K61, in Mycobacterium smegmatis Pup, K7 and K31 are involved in the formation of the poly-Pup chain in PafA poly-pupylation. Poly-pupylation of PafA can be reversibly regulated by depupylase Dop Mycolicibacterium smegmatis

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
ATP + [prokaryotic ubiquitin-like protein]-L-glutamate + [PanB]-L-lysine
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Mycolicibacterium smegmatis ADP + phosphate + N6-([prokaryotic ubiquitin-like protein]-gamma-L-glutamyl)-[PanB]-L-lysine
-
?
ATP + [prokaryotic ubiquitin-like protein]-L-glutamate + [PanB]-L-lysine
-
Mycolicibacterium smegmatis ATCC 700084 / mc2155 ADP + phosphate + N6-([prokaryotic ubiquitin-like protein]-gamma-L-glutamyl)-[PanB]-L-lysine
-
?
ATP + [prokaryotic ubiquitin-like protein]-L-glutamate + [protein]-L-lysine
-
Mycolicibacterium smegmatis ADP + phosphate + N6-([prokaryotic ubiquitin-like protein]-gamma-L-glutamyl)-[protein]-L-lysine
-
?
ATP + [prokaryotic ubiquitin-like protein]-L-glutamate + [protein]-L-lysine
-
Mycolicibacterium smegmatis ATCC 700084 / mc2155 ADP + phosphate + N6-([prokaryotic ubiquitin-like protein]-gamma-L-glutamyl)-[protein]-L-lysine
-
?
additional information PafA can catalyze the poly-pupylation of itself, poly-pupylation of PafA. One common pupylation substrate is protein PanB. PafA self-pupylation is suppressed when PanB is added at high concentrations. Unlike the pattern of poly-pupylated PafA, only mono-pupylated PanB and a few di-pupylated PanB (Pup2-PanB) can be detected in the reaction system Mycolicibacterium smegmatis ?
-
?
additional information PafA can catalyze the poly-pupylation of itself, poly-pupylation of PafA. One common pupylation substrate is protein PanB. PafA self-pupylation is suppressed when PanB is added at high concentrations. Unlike the pattern of poly-pupylated PafA, only mono-pupylated PanB and a few di-pupylated PanB (Pup2-PanB) can be detected in the reaction system Mycolicibacterium smegmatis ATCC 700084 / mc2155 ?
-
?

Synonyms

Synonyms Comment Organism
PafA
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Mycolicibacterium smegmatis
Pup ligase
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Mycolicibacterium smegmatis

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
23
-
assay at Mycolicibacterium smegmatis

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
assay at Mycolicibacterium smegmatis

Cofactor

Cofactor Comment Organism Structure
ATP
-
Mycolicibacterium smegmatis

General Information

General Information Comment Organism
physiological function enzyme PafA, the prokaryotic ubiquitin-like protein (Pup) ligase, catalyzes the Pup modification of bacterial proteins and targets the substrates for proteasomal degradation. Mycobacterium smegmatis PafA can be poly-pupylated. Self-pupylation of PafA is reversely regulated by Dop, a dual-functional enzyme, functioning as deaminase to convert PupQ to PupE and also as depupylase to remove Pup from the pupylated proteins. The self-pupylation of PafA is involved in the regulation of its stability Mycolicibacterium smegmatis