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 | 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 | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | required | Mycolicibacterium smegmatis |
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 | - |
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 | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Mycolicibacterium smegmatis | A0QZ42 | - |
- |
Mycolicibacterium smegmatis ATCC 700084 / mc2155 | A0QZ42 | - |
- |
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 | 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 | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + [prokaryotic ubiquitin-like protein]-L-glutamate + [PanB]-L-lysine | - |
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 | Comment | Organism |
---|---|---|
PafA | - |
Mycolicibacterium smegmatis |
Pup ligase | - |
Mycolicibacterium smegmatis |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
23 | - |
assay at | Mycolicibacterium smegmatis |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
assay at | Mycolicibacterium smegmatis |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
ATP | - |
Mycolicibacterium smegmatis |
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 |