Information on EC 6.3.1.19 - prokaryotic ubiquitin-like protein ligase

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The expected taxonomic range for this enzyme is: Bacteria, Eukaryota

EC NUMBER
COMMENTARY hide
6.3.1.19
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RECOMMENDED NAME
GeneOntology No.
prokaryotic ubiquitin-like protein ligase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + [prokaryotic ubiquitin-like protein]-L-glutamate + [protein]-L-lysine = ADP + phosphate + N6-([prokaryotic ubiquitin-like protein]-gamma-L-glutamyl)-[protein]-L-lysine
show the reaction diagram
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
protein Pupylation and dePupylation
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SYSTEMATIC NAME
IUBMB Comments
[prokaryotic ubiquitin-like protein]:[protein]-L-lysine
The enzyme has been characterized from the bacteria Mycobacterium tuberculosis and Corynebacterium glutamicum. It catalyses the ligation of the prokaryotic ubiquitin-like protein (Pup) to a target protein by forming a bond between an epsilon-amino group of a lysine residue of the target protein and the gamma-carboxylate of the C-terminal glutamate of the ubiquitin-like protein (Pup). The attachment of Pup, also known as Pupylation, marks proteins for proteasomal degradation.
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
Acidothermus cellulolyticus ATCC 43068 / 11B
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UniProt
Manually annotated by BRENDA team
Bifidobacterium adolescentis ATCC 15703 / DSM 20083 / NCTC 11814 / E194a
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UniProt
Manually annotated by BRENDA team
Corynebacterium glutamicum ATCC 13032 / DSM 20300 / JCM 1318 / LMG 3730 / NCIMB 10025
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SwissProt
Manually annotated by BRENDA team
-
UniProt
Manually annotated by BRENDA team
Kocuria rhizophila ATCC 9341 / DSM 348 / NBRC 103217 / DC2201
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UniProt
Manually annotated by BRENDA team
i.e. Micrococcus lysodeikticus
UniProt
Manually annotated by BRENDA team
Micrococcus luteus ATCC 4698 / DSM 20030 / JCM 1464 / NBRC 3333 / NCIMB 9278 / NCTC 2665 / VKM Ac-2230
i.e. Micrococcus lysodeikticus
UniProt
Manually annotated by BRENDA team
Mycobacterium smegmatis ATCC 700084 / mc2155
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UniProt
Manually annotated by BRENDA team
Mycobacterium tuberculosis ATCC 25618 / H37Rv
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SwissProt
Manually annotated by BRENDA team
i.e. Arthrobacter aurescens
UniProt
Manually annotated by BRENDA team
i.e. Arthrobacter aurescens
UniProt
Manually annotated by BRENDA team
Renibacterium salmoninarum ATCC 33209 / DSM 20767 / JCM 11484 / NBRC 15589 / NCIMB 2235
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UniProt
Manually annotated by BRENDA team
Rhodococcus erythropolis PR4 / NBRC 100887
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UniProt
Manually annotated by BRENDA team
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UniProt
Manually annotated by BRENDA team
Salinispora tropica ATCC BAA-916 / DSM 44818 / CNB-440
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UniProt
Manually annotated by BRENDA team
Streptomyces coelicolor ATCC BAA-471 / A3(2) / M145
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UniProt
Manually annotated by BRENDA team
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UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
malfunction
metabolism
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ATP + [prokaryotic ubiquitin-like protein]-L-glutamate + [adenylate kinase-His6]-L-lysine
ADP + phosphate + N6-([prokaryotic ubiquitin-like protein]-gamma-L-glutamyl)-[adenylate kinase-His6]-L-lysine
show the reaction diagram
ATP + [prokaryotic ubiquitin-like protein]-L-glutamate + [FabD]-L-lysine
ADP + phosphate + N6-([prokaryotic ubiquitin-like protein]-gamma-L-glutamyl)-[FabD]-L-lysine
show the reaction diagram
ATP + [prokaryotic ubiquitin-like protein]-L-glutamate + [IdeR]-L-lysine
ADP + phosphate + N6-([prokaryotic ubiquitin-like protein]-gamma-L-glutamyl)-[IdeR]-L-lysine
show the reaction diagram
ATP + [prokaryotic ubiquitin-like protein]-L-glutamate + [inositol 1-phosphate synthetase]-L-lysine
ADP + phosphate + N6-([prokaryotic ubiquitin-like protein]-gamma-L-glutamyl)-[inositol 1-phosphate synthetase]-L-lysine
show the reaction diagram
ATP + [prokaryotic ubiquitin-like protein]-L-glutamate + [Log]-L-lysine
ADP + phosphate + N6-([prokaryotic ubiquitin-like protein]-gamma-L-glutamyl)-[Log]-L-lysine
show the reaction diagram
ATP + [prokaryotic ubiquitin-like protein]-L-glutamate + [malonyl Co-A acyl carrier protein transacylase]-L-lysine
ADP + phosphate + N6-([prokaryotic ubiquitin-like protein]-gamma-L-glutamyl)-[malonyl Co-A acyl carrier protein transacylase]-L-lysine
show the reaction diagram
ATP + [prokaryotic ubiquitin-like protein]-L-glutamate + [myo-inositol-1-phosphate synthetase]-L-lysine
ADP + phosphate + N6-([prokaryotic ubiquitin-like protein]-gamma-L-glutamyl)-[myo-inositol-1-phosphate synthetase]-L-lysine
show the reaction diagram
ATP + [prokaryotic ubiquitin-like protein]-L-glutamate + [PanB]-L-lysine
ADP + phosphate + N6-([prokaryotic ubiquitin-like protein]-gamma-L-glutamyl)-[PanB]-L-lysine
show the reaction diagram
ATP + [prokaryotic ubiquitin-like protein]-L-glutamate + [phosphoenolpyruvate-protein phosphotransferase I]-L-lysine
ADP + phosphate + N6-([prokaryotic ubiquitin-like protein]-gamma-L-glutamyl)-[phosphoenolpyruvate-protein phosphotransferase I]-L-lysine
show the reaction diagram
ATP + [prokaryotic ubiquitin-like protein]-L-glutamate + [prokaryotic ubiquitin-like protein]-L-lysine
ADP + phosphate + N6-([prokaryotic ubiquitin-like protein]-gamma-L-glutamyl)-[prokaryotic ubiquitin-like protein]-L-lysine
show the reaction diagram
in addition to the lysine at position 61, Pup (prokaryotic ubiquitin-like protein) presents two more lysines, one at position 7 and another at position 31. Pup can be pupylated on different lysines
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?
ATP + [prokaryotic ubiquitin-like protein]-L-glutamate + [proteasome-interacting ATPase]-L-lysine
ADP + phosphate + N6-([prokaryotic ubiquitin-like protein]-gamma-L-glutamyl)-[proteasome-interacting ATPase]-L-lysine
show the reaction diagram
ATP + [prokaryotic ubiquitin-like protein]-L-glutamate + [protein PanB]-L-lysine
ADP + phosphate + N6-([prokaryotic ubiquitin-like protein]-gamma-L-glutamyl)-[protein PanB]-L-lysine
show the reaction diagram
ATP + [prokaryotic ubiquitin-like protein]-L-glutamate + [protein]-L-lysine
ADP + phosphate + N6-([prokaryotic ubiquitin-like protein]-gamma-L-glutamyl)-[protein]-L-lysine
show the reaction diagram
ATP + [prokaryotic ubiquitin-like protein]-L-glutamate + [protein]-L-lysine
ADP + phosphate + N6-[[prokaryotic ubiquitin-like protein]-L-glutamate]-[[protein]-L-lysine]
show the reaction diagram
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
ATP + [prokaryotic ubiquitin-like protein]-L-glutamate + [proteasome-interacting ATPase]-L-lysine
ADP + phosphate + N6-([prokaryotic ubiquitin-like protein]-gamma-L-glutamyl)-[proteasome-interacting ATPase]-L-lysine
show the reaction diagram
ATP + [prokaryotic ubiquitin-like protein]-L-glutamate + [protein]-L-lysine
ADP + phosphate + N6-([prokaryotic ubiquitin-like protein]-gamma-L-glutamyl)-[protein]-L-lysine
show the reaction diagram
ATP + [prokaryotic ubiquitin-like protein]-L-glutamate + [protein]-L-lysine
ADP + phosphate + N6-[[prokaryotic ubiquitin-like protein]-L-glutamate]-[[protein]-L-lysine]
show the reaction diagram
additional information
?
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0087
ATP
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25°C, pH 7.4
0.22
[IdeR]-L-lysine
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pH 7.5, 30°C, recombinant His-tagged enzyme
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0.084
[PanB]-L-lysine
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pH 7.5, 30°C, recombinant His-tagged enzyme
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0.0014
[prokaryotic ubiquitin-like protein]-L-glutamate
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25°C, pH 7.4
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0.0142
[protein PanB]-L-lysine
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25°C, pH 7.4
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additional information
additional information
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.014
[IdeR]-L-lysine
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pH 7.5, 30°C, recombinant His-tagged enzyme
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0.0023
[prokaryotic ubiquitin-like protein]-L-glutamate
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pH 8.0, 30°C
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0.023
[protein PanB]-L-lysine
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pH 8,0, 30°C
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
PDB
SCOP
CATH
UNIPROT
ORGANISM
Corynebacterium glutamicum (strain ATCC 13032 / DSM 20300 / JCM 1318 / LMG 3730 / NCIMB 10025);
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystal structure of the enzyme with ADP, protein (Pup) ligase PafA and the depupylase/deamidase Dop are are close structural homologues
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the enzyme from Corynebacterium glutamicum is C-terminally fused with Pup or N-terminally truncated fragments of prokaryotic ubiquitin-like protein (Pup) from the same organism. Crystals are obtained only with the shortest fragment tested, PupE38-64. The crystal structure of Pup in complex with the enzyme, reveals that a long groove wrapping around the enzyme serves as a docking site for Pup. Upon binding, the C-terminal region of the intrinsically disordered Pup becomes ordered to form two helices connected by a linker, positioning the C-terminal glutamate in the active site of PafA
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant enzyme expressed in Escherichia coli
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recombinant His6-tagged enzyme expressed in Escherichia coli
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recombinant His6-tagged enzyme from Escherichia coli by nickel affinity chormatography
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recombinant His6-tagged enzyme from Escherichia coli strain ER2566 by nickel affinity chormatography
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
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
gene pafA, genetic arrangement in the pupylation gene locus, comparisons, overview
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
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recombinant expression of His6-tagged enzyme in Escherichia coli
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recombinant expression of His6-tagged enzyme in Escherichia coli strain ER2566
reconstitution of the Mycobacterium tuberculosis pupylation pathway in Escherichia coli
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the pafA gene is cloned with a C-terminal His6-tag, expression in Escherichia coli
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A196S
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site-directed mutagenesis, the mutant shows increased pupylation activity with substrate PanB compared to wild-type
D208A
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site-directed mutagenesis, the mutant shows reduced pupylation activity with substrate PanB compared to wild-type
D64N
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site-directed mutagenesis, a catalytically inactive mutant
E209A
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site-directed mutagenesis, the mutant shows highly reduced pupylation activity with substrate PanB compared to wild-type
E70A
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site-directed mutagenesis, a catalytically inactive mutant
N205A
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site-directed mutagenesis, the mutant shows reduced pupylation activity with substrate PanB compared to wild-type
R199A
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site-directed mutagenesis, the mutant shows highly reduced pupylation activity with substrate PanB compared to wild-type
R201A
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site-directed mutagenesis, pupylation catalytically inactive mutant
R207A
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site-directed mutagenesis, the mutant shows highly reduced pupylation activity with substrate PanB compared to wild-type; 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
T197A
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site-directed mutagenesis, the mutant shows reduced pupylation activity with substrate PanB compared to wild-type
T198A
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site-directed mutagenesis, the mutant shows highly reduced pupylation activity with substrate PanB compared to wild-type
V65S
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site-directed mutagenesis, the mutant shows reduced pupylation activity with substrate PanB compared to wild-type
D208A
Corynebacterium glutamicum ATCC 13032 / DSM 20300 / JCM 1318 / LMG 3730 / NCIMB 10025
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site-directed mutagenesis, the mutant shows reduced pupylation activity with substrate PanB compared to wild-type
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D64N
Corynebacterium glutamicum ATCC 13032 / DSM 20300 / JCM 1318 / LMG 3730 / NCIMB 10025
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site-directed mutagenesis, a catalytically inactive mutant
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R201A
Corynebacterium glutamicum ATCC 13032 / DSM 20300 / JCM 1318 / LMG 3730 / NCIMB 10025
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site-directed mutagenesis, pupylation catalytically inactive mutant
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T197A
Corynebacterium glutamicum ATCC 13032 / DSM 20300 / JCM 1318 / LMG 3730 / NCIMB 10025
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site-directed mutagenesis, the mutant shows reduced pupylation activity with substrate PanB compared to wild-type
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T198A
Corynebacterium glutamicum ATCC 13032 / DSM 20300 / JCM 1318 / LMG 3730 / NCIMB 10025
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site-directed mutagenesis, the mutant shows highly reduced pupylation activity with substrate PanB compared to wild-type
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E9A
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site-directed mutagenesis, the PafA mutant without ligase activity cannot be pupylated
K31R
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site-directed mutagenesis, the mutation attenuates the poly-pupylation of PafA
K320R
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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
K61R
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site-directed mutagenesis, the mutation attenuates the poly-pupylation of PafA
K7R
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site-directed mutagenesis, the mutation attenuates the poly-pupylation of PafA
E9A
Mycobacterium smegmatis ATCC 700084 / mc2155
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site-directed mutagenesis, the PafA mutant without ligase activity cannot be pupylated
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K320R
Mycobacterium smegmatis ATCC 700084 / mc2155
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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
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K61R
Mycobacterium smegmatis ATCC 700084 / mc2155
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site-directed mutagenesis, the mutation attenuates the poly-pupylation of PafA
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K7R
Mycobacterium smegmatis ATCC 700084 / mc2155
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site-directed mutagenesis, the mutation attenuates the poly-pupylation of PafA
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additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
drug development
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