Information on EC 6.2.1.45 - E1 ubiquitin-activating enzyme

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

EC NUMBER
COMMENTARY hide
6.2.1.45
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RECOMMENDED NAME
GeneOntology No.
E1 ubiquitin-activating enzyme
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + ubiquitin + [E1 ubiquitin-activating enzyme]-L-cysteine = AMP + diphosphate + S-ubiquitinyl-[E1 ubiquitin-activating enzyme]-L-cysteine
show the reaction diagram
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
protein ubiquitylation
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SYSTEMATIC NAME
IUBMB Comments
ubiquitin:[E1 ubiquitin-activating enzyme] ligase (AMP-forming)
Catalyses the ATP-dependent activation of ubiquitin through the formation of a thioester bond between the C-terminal glycine of ubiquitin and the sulfhydryl side group of a cysteine residue in the E1 protein. The two-step reaction consists of the ATP-dependent formation of an E1-ubiquitin adenylate intermediate in which the C-terminal glycine of ubiquitin is bound to AMP via an acyl-phosphate linkage, then followed by the conversion to an E1-ubiquitin thioester bond via the cysteine residue on E1 in the second step.
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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Manually annotated by BRENDA team
cv. Wuzishatangju (seedless) and cv. Shatangju (seedy), single copy gene CrUBE1
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
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5 amino acids and 8 bases are different in cDNA and DNA sequences of CrUBE1 between Wuzishatangju and Shatangju, respectively
malfunction
metabolism
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ATP + Oregon Green-labeled ubiquitin + [ubiquitin-activating protein E1]-L-cysteine
AMP + diphosphate + [ubiquitin-activating protein E1]-S-(Oregon Green-labeled ubiquitinyl)-L-cysteine
show the reaction diagram
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enzyme efficiently accepts ubiquitin substrate fluorescently labeled by Oregon Green
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-
?
ATP + SUMO2 + [ubiquitin-activating protein UBA5]-L-cysteine
AMP + diphosphate + [ubiquitin-activating protein UBA5]-S-SUMO2-L-cysteine
show the reaction diagram
SUMO2, small ubiquitin-like modifier2, an ubiquitin-like protein
enzyme greatly activates SUMO2 in the nucleus or transfers activated SUMO2 to the nucleus after it conjugated SUMO2 in the cytoplasm
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?
ATP + ubiquitin + SUMO2
?
show the reaction diagram
UBE1DC1 greatly activates SUMO2 in the nucleus or transfers activated-SUMO2 to nucleus after conjugation of SUMO2 in the cytoplasm
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-
?
ATP + ubiquitin + ubiquitin carrier protein E2
AMP + diphosphate + ubiquitin-(ubiquitin carrier protein E2)
show the reaction diagram
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-
-
-
?
ATP + ubiquitin + ubiquitin-fold modifier 1
?
show the reaction diagram
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-
-
?
ATP + ubiquitin + Ufm1
?
show the reaction diagram
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-
-
?
ATP + ubiquitin + [6His-ubiquitin-activating enzyme E1]W-8His-Strep-HA
AMP + diphosphate + [6His-ubiquitin-activating enzyme E1]W-8His-Strep-HA-ubiquitinyl-L-cysteine
show the reaction diagram
Strep, i.e.WSHPQFEK, HA, i.e. YPYDVPDYAS, under non-reducing conditions, the intermediate complex of the thioester formation is not observed without ATP
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-
?
ATP + ubiquitin + [E1 ubiquitin-activating enzyme]-L-cysteine
AMP + diphosphate + S-ubiquitinyl-[E1 ubiquitin-activating enzyme]-L-cysteine
show the reaction diagram
ATP + ubiquitin + [ubiquitin-activating enzyme Uba5]-L-cysteine
AMP + diphosphate + [ubiquitin-activating enzyme Uba5]-S-ubiquitinyl-L-cysteine
show the reaction diagram
the catalytic cysteine residue of isoform Uba5 is part of the adenylation domain in a alpha-helical motif
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ATP + ubiquitin + [ubiquitin-activating protein E1]-L-cysteine
AMP + diphosphate + [ubiquitin-activating protein E1]-S-ubiquitinyl-L-cysteine
show the reaction diagram
ATP + ubiquitin + [ubiquitin-activating protein Uba1a]-L-cysteine
AMP + diphosphate + [ubiquitin-activating protein Uba1a]-S-ubiquitinyl-L-cysteine
show the reaction diagram
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-
-
-
?
ATP + ubiquitin + [ubiquitin-activating protein UBA1]-L-cysteine
AMP + diphosphate + [ubiquitin-activating protein UBA1]-S-ubiquitinyl-L-cysteine
show the reaction diagram
ATP + ubiquitin + [ubiquitin-activating protein UBA2]-L-cysteine
AMP + diphosphate + [ubiquitin-activating protein UBA2]-S-ubiquitinyl-L-cysteine
show the reaction diagram
enzyme forms higher molecular mass intermediates with ubiquitin
the enzyme-ubiquitin intermediates dissociate in presence of 2-mercaptoethanol, indicating thiolester linkage
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?
ATP + ubiquitin + [ubiquitin-activating protein Uba3]-L-cysteine
AMP + diphosphate + [ubiquitin-activating protein Uba3]-S-ubiquitinyl-L-cysteine
show the reaction diagram
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-
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ATP + ubiquitin + [ubiquitin-activating protein Uba6]-L-cysteine
AMP + diphosphate + [ubiquitin-activating protein Uba6]-S-ubiquitinyl-L-cysteine
show the reaction diagram
isoform Uba6 forms a covalent link with ubiquitin in vitro and in vivo, which is sensitive to reducing conditions. Recombinant E1 enzyme Uba6 can activate ubiquitin and transfer it onto the ubiquitin-conjugating enzyme UbcH5B. Ubiquitin activated by Uba6 can be used for ubiquitylation of p53 and supports the autoubiquitylation of the E3 ubiquitin ligases HectH9 and E6-AP
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-
?
ATP + ubiquitin + [ubiquitin-activating protein UBE1]-L-cysteine
AMP + diphosphate + [ubiquitin-activating protein UBE1]-S-ubiquitinyl-L-cysteine
show the reaction diagram
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-
-
?
ATP + ubiquitin fold modifier1 + [ubiquitin-activating enzyme Uba5]-L-cysteine
AMP + diphosphate + [ubiquitin-activating enzyme Uba5]-S-(ubiquitin fold modifier1)-L-cysteine
show the reaction diagram
the catalytic cysteine residue of isoform Uba5 is part of the adenylation domain in a alpha-helical motif
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ATP + ubiquitin mutant G76A + [ubiquitin-activating protein E1]-L-cysteine
AMP + diphosphate + [ubiquitin-activating protein E1]-S-(ubiquitin mutant G76A)yl-L-cysteine
show the reaction diagram
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mutant ubiquitin G76A, bearing a Gly to Ala substitution at the COOH terminus is a substrate for El enzyme. Ubiquitin G76A supports PPI-ATP exchange with 500fold decrease in kcat/Km compared to wild-type ubiquitin, does not produce detectable AMP-Ub with native El, produces stoichiometric AMP-Ub with thiol-blocked El, gives a stoichiometric burst of ATP consumption with either native or thiol-blocked El, support El-ubiquitin thiol ester formation with native El, and supports several downstream reactions of the proteolytic pathway with a 20% decrease to the rate of wild type ubiquitin
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?
ATP + Ufm1 + [ubiquitin-activating protein UBA5]-L-cysteine
AMP + diphosphate + [ubiquitin-activating protein UBA5]-S-Ufm1-L-cysteine
show the reaction diagram
Ufm1, ubiquitin-fold modifier 1, an ubiquitin-like protein
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?
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 + ubiquitin + [E1 ubiquitin-activating enzyme]-L-cysteine
AMP + diphosphate + S-ubiquitinyl-[E1 ubiquitin-activating enzyme]-L-cysteine
show the reaction diagram
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Zinc
isoform UBA5 maintains a zinc-binding site that is coordinated by four cysteines with tetrahedral geometry
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(3E)-4-[(5R,8S)-5-methyl-6,9,13-trioxo-8-(propan-2-yl)-10-oxa-3,17-dithia-7,14,19,20-tetraazatricyclo[14.2.1.1~2,5~]icosa-1(18),2(20),16(19)-trien-11-yl]but-3-en-1-yl octanoate
(5R,8S)-11-ethenyl-5-methyl-8-(propan-2-yl)-10-oxa-3,17-dithia-7,14,19,20-tetraazatricyclo[14.2.1.1~2,5~]icosa-1(18),2(20),16(19)-triene-6,9,13-trione
(5R,8S)-5-methyl-11-[(1E)-6-oxotridec-1-en-1-yl]-8-(propan-2-yl)-10-oxa-3,17-dithia-7,14,19,20-tetraazatricyclo[14.2.1.1~2,5~]icosa-1(18),2(20),16(19)-triene-6,9,13-trione
(5R,8S)-5-methyl-8-(propan-2-yl)-11-[(1E)-4-sulfanylbut-1-en-1-yl]-10-oxa-3,17-dithia-7,14,19,20-tetraazatricyclo[14.2.1.1~2,5~]icosa-1(18),2(20),16(19)-triene-6,9,13-trione
1-(3-chloro-4-fluorophenyl)-4-[(5-nitro-2-furyl)methylene]-3,5-pyrazolidinedione
4[4-(5-nitro-furan-2-ylmethylene)-3,5-dioxo-pyrazolidin-1-yl]-benzoic acid ethyl ester
5'-[[(L-cysteinylglycylglycyl)sulfamoyl]amino]-5'-deoxyadenosine
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inhibits Uba1-S-Ub thioester formation in a dose-dependent manner. The inhibitor is highly selective for its cognate E1 enzyme and does not inhibit the corresponding non-cognate E1s
ginsenoside Re
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inhibits ubiquitin-activating enzyme, from Panax ginseng roots, a traditional herbal medicine or food
ginsenoside Rg1
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inhibit ubiquitin-activating enzyme, from Panax ginseng roots, a traditional herbal medicine or food, causes 89.2% inhibition at 0.05 mM
largazole
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largazole and its ester and ketone analogues selectively inhibit human UBA1 enzyme and inhibit ubiquitin conjugation to cyclin-dependent kinase inhibitor p27Kip1 and TRF1 in vitro, mechanism of E1 inhibition, overview. Largazole and its derivatives specifically inhibit the adenylation step of the E1 reaction while having no effect on thioester bond formation between ubiquitin and E1. Upon incubation with E1, largazole or largazole ester reduce the amount of ubiquitin molecules that are transferred from E1 to E2 in a dose-dependent fashion. E1 inhibition appears to be specific to human E1. Largazole analogues do not significantly inhibit SUMO E1
S-[(3E)-5-hydroxy-7-({[(4R)-4-{[(3S)-2-methoxy-4-methylpent-1-en-3-yl]carbamoyl}-4-methyl[4,5-dihydro[2,4'-bi-1,3-thiazole]]-2'-yl]methyl}amino)-7-oxohept-3-en-1-yl] octanethioate
trichostatin A
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[ubiquitin carrier protein Ubc4]-L-cysteine
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additional information
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largazole and its ester and ketone analogues selectively inhibit ubiquitin conjugation to p27Kip1 and TRF1 in vitro, but the inhibition appears to be specific to human E1. Largazole analogues do not significantly inhibit Uba1p
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
ginsenoside Rb1
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increases ubiquitination on E1 enzyme, from Panax ginseng roots, a traditional herbal medicine or food
ginsenoside Rb2
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increases ubiquitination on E1 enzyme, from Panax ginseng roots, a traditional herbal medicine or food
ginsenoside Rc
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increases ubiquitination on E1 enzyme, from Panax ginseng roots, a traditional herbal medicine or food
ginsenoside Rd
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increases ubiquitination on E1 enzyme, from Panax ginseng roots, a traditional herbal medicine or food
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0045 - 0.208
ATP
0.00017
Oregon Green-labeled ubiquitin
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pH 7.5, 25°C
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0.00017 - 0.029
ubiquitin
0.000073 - 0.000135
ubiquitin carrier protein E2
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.000075 - 6
ATP
0.005 - 6
ubiquitin
0.005 - 6
ubiquitin carrier protein E2
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0035
[ubiquitin carrier protein Ubc4]-L-cysteine
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pH 7.5, 25°C
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IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0628
ginsenoside Re
Homo sapiens
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recombinant enzyme, pH 7.6, 37°C
0.0035
ginsenoside Rg1
Homo sapiens
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recombinant enzyme, pH 7.6, 37°C
additional information
4[4-(5-nitro-furan-2-ylmethylene)-3,5-dioxo-pyrazolidin-1-yl]-benzoic acid ethyl ester
Homo sapiens
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inhibitor blocks loading of immobilized His6-tagged E1 with ubiquitin with an IC50 below 10 microM
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.2
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assay at
7.6
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assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.44
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sequence calculation
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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mouse embryo fibroblast cell, thermosensitive for ubiquitin-activating enzyme E1
Manually annotated by BRENDA team
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expression level of the CrUBE1 gene in anthers of cv. Shatangju is approximately 10fold higher than in anthers of cv. Wuzishatangju
Manually annotated by BRENDA team
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high enzyme expression level
Manually annotated by BRENDA team
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very low expression
Manually annotated by BRENDA team
embryonic kidney
Manually annotated by BRENDA team
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very low expression
Manually annotated by BRENDA team
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very low expression
Manually annotated by BRENDA team
colon carcinoma cell
Manually annotated by BRENDA team
additional information
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
predominant localization upon cotransfection with small ubiquitin-like modifier SUMO2; UBE1DC1 is mainly distributed in the nucleus of AD-293 cells when cotransfected with its substrate SUMO2
Manually annotated by BRENDA team
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Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
UNIPROT
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Schizosaccharomyces pombe (strain 972 / ATCC 24843)
Schizosaccharomyces pombe (strain 972 / ATCC 24843)
Schizosaccharomyces pombe (strain 972 / ATCC 24843)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
12000
x * 12000 and x * 124000, SDS-PAGE, x * 119600, calculated
42000
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x * 42000, recombinant mRFP-enzyme, SDS-PAGE
47000
1 * 68000 + 1 * 47000, the enzyme is proteolytically processed mainly into two fragments of 68 kDa (N-terminal) and 47 kDa (C-terminal)
49280
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x * 49280, sequence calculation
68000
1 * 68000 + 1 * 47000, the enzyme is proteolytically processed mainly into two fragments of 68 kDa (N-terminal) and 47 kDa (C-terminal)
110450
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x * 110450, calculated
112000
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analytical ultracentrifugation
114000
complete E1 protein, native PAGE
117000
x * 117000, SDS-PAGE
117300
-
1 * 117300, calculated
118000
SDS-PAGE
119600
x * 12000 and x * 124000, SDS-PAGE, x * 119600, calculated
120300
x * 123000, SDS-PAGE, x * 120300, calculated
123000
x * 123000, SDS-PAGE, x * 120300, calculated
124000
x * 12000 and x * 124000, SDS-PAGE, x * 119600, calculated
130000
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recombinant His6-tagged chimeric mutant Aos1-Uba2 SUMO-E1 enzyme mAU, gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
additional information
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the enzyme N-terminal domains contain inactive adenylation domain (IAD) and the first catalytic cysteine half-domain (FCCH). The IAD domain covers from Met1 to Glu204 and Val295 to Ile439 and that the FCCH domain covers from Glu205 to Gln294. The structure of ubiquitin E1 consists of four different domain blocks, overview. The enzyme forms a pseudo-dimer
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
proteolytic modification
additional information
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conjugation of the ubiquitin activating enzyme UBE1 with the ubiquitin-like modifier FAT10 targets it for proteasomal degradation
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
enzyme exists in two splice variants. To obtain high resolution crystals of UBA5, the N-terminal region of the long isoform, residues 1–56, are deleted and residues 330–404 of the C-terminal domain are also removed. The removal of the CTD thus does not abrogate formation of the UBA5-UFM1 thioester intermediate. Crystals to 2.0 A resolution, and molecular replacement based on PDB structure 1ZFN. Structure shows similarities to both E1 and E1-like enzymes and is composed of an ATP-binding domain that consists of an eight-stranded beta-sheet surrounded by seven alpha-helices. UBA5 maintains a zinc-binding site that is coordinated by four cysteines with tetrahedral geometry; to 2.0 A resolution using molecular replacement based on PDB entry 1ZFN. UBA5 structure shows similarities to both E1 and E1-like enzymes and is composed of an ATP-binding domain that consists of an eight-stranded beta-sheet surrounded by seven alpha-helices. UBA5 maintains a zinc-binding site that is coordinated by four cysteines with tetrahedral geometry
molecular modelling based on the crystal structure of Saccharomyces cerevisiae E1 and Mus musculus E1 and molecular dynamics simulation in water of the human E1-Ub complex
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purified recombinant N-terminal enzyme domains comprising residues 1-439, hanging drop vapor diffusion method, mixing of 0.0015 ml of 15 mg/ml protein in 10mM Tris-HCl, pH 8.0, 150mM NaCl, and 2 mM DTT, with 0.0015 ml of reservoir solution containing 0.1 M Na3-citrate, pH 5.6, and 3.2 M NH4Ac, microseeding, 3 days, 21°C, X-ray diffraction structure determination and analysis at 2.75 A resolution
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NMR structural studies of the first catalytic cysteine half domain FCCH, interaction studies of FCCH and the other catalytic E1 domain SCCH, second catalytic cysteine half-domain. The E1 has several domains, an adenylation domain, composed of an active and inactive adenylation subdomains, and a catalytic cysteine domain, and smaller accessory domains: a four helix bundle and a ubiquitin fold domain. NMR cannot detect interactions between the FCCH and ubiquitin, or betweenween FCCH and SCCH if they are on separate poypeptide chains
crystal structures of the C-terminal ubiquitin fold domain from yeast Uba2 alone and in complex with E2 enzyme Ubc9. Uba2 undergoes remarkable conformational changes during the reaction. The structure of the Uba2 domain-Ubc9 complex reveals interactions unique to Sumo E1 and E2. Comparison with a previous Ubc9-E3 complex structure demonstrates overlap between Uba2 and E3 binding sites on Ubc9, indicating that loading with Sumo and E3-catalyzed transfer to substrates are strictly separate steps
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-70°C, recombinant enzyme, no detectable loss of activity
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-70°C, the storage of the recombinant mouse E1 does not result in a detectable denaturation of the enzyme
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
His6-tagged E1 expressed and purified in Saccharomyces cerevisiae MHY-501 cells
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Ni-NTA column chromatography
recombinant enzyme, purification on Ni-NTA superflow sepharose and strep-tactin sepharose which is based on UB-UBE1 high-energy thioester bonded intermediate complex; recombinant protein; recombinant protein is purified by Ni-NTA His-Bind Superflow Sepharose and Strep-Tactin Sepahrose
recombinant GST-tagged enzyme from Hi5 insect cells by glutathione affinity chromatography, tag cleavage by thrombin, and gel filtration
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recombinant His-tagged chimeric mutant Aos1-Uba2 SUMO-E1 enzyme mAU from insect cells by nickel affinity chromatography
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recombinant His-tagged N-terminal enzyme domains from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, anion exchange chromatography, tag cleavage by 3C protease, another step of nickel affinity chromatography, followed by gel filtration
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recombinant mRFP-enzyme from Escherichia coli strain DH5alpha by combined anion exchange/affinity chromatography and gel filtration
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recombinant protein, HIS-Select nickel affinity chromatography, further steps, ion exchange chromatography, followed by gel filtration
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
; expression in BL-21 cell
expresion in Escherichia coli; expressed in Escherichia coli BL21(DE3) using pET28-mE1 plasmid
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expressed in Escherichia coli BL21(DE3) cells
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expressed in Escherichia coli Rosetta cells and in AD-293 cells
expression in baculoviral system
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expression in Escherichia coli; expression in Escherichia coli
expression in Escherichia coli; expression in Escherichia coli; full length of human UBE1 is expressed in Escherichia coli via pET28b vector
gene CrUBE1, genotyping, DNA and amino acid sequence determination and analysis, sequence comparisons, semi-quantitative RT-PCR and quantitative real-time PCR enzyme expression anayses, recombinant expression in Pichia pastoris strain GS115
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genotyping-phenotyping
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HEK293 cells are transiently co-transfected with expression plasmids for HA-UBE1, the HA-tagged active site cysteine mutant UBE1 C632A, a His3-FLAG-tagged FAT10, a His3-FLAG-tagged FAT10 mutant lacking a diglycine motif at the C-terminus, or a His3-FLAG-tagged lysine-less FAT10 mutant
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His6-tagged E1 expressed and purified in Saccharomyces cerevisiae MHY-501 cells
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overexpression of N-terminally or C-terminally HA-tagged enzyme in Giardia intestinalis trophozoites
recombinant baculovirus-mediated expression of the GST-tagged enzyme in Hi5 insect cells using the pFastBacHTA vector
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recombinant expression of His6-tagged chimeric mutant Aos1-Uba2 SUMO-E1 enzyme mAU in Spodoptera frugiperda Sf9 insect cells via baculovirus transformation, mAU has SUMO-E1 activity. Recombinant expression of GST-tagged mAU in Escherichia coli
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recombinant expression of mRFP-enzyme in Escherichia coli strain DH5alpha, linking of ubiquitin to the C-terminus of RFP through a peptide bond
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recombinant expression of N-terminally His6-tagged N-terminal domains of the enzyme, residues 1-439, in Escherichia coli strain BL21(DE3) from vector pETDuet-1
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
self pollination induces the enzyme in cv. Wuzishatangju
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the enzyme expression (both at mRNA and protein levels) is regulated during encystation
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C632A
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generation of an active site cysteine mutant of HA-UBE1
D576A
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Km-value for ATP is 37.8fold higher than wild-type value, KM-value for ubiquitin is 36fold higher than wild-type value. kcat for ubiquitin adenylate formation is 250fold lower than wild-type value. kcat for ubiquitin carrier protein E2 transthiolation is 28.3fold lower than wild-type value; mutation within the MgATP2- binding site, results in dramatically impaired binding affinities for MgATP2-, a shift from ordered to random addition in co-substrate binding, and a significantly reduced rate of ternary complex formation that shifts the rate-limiting step to ubiquitin adenylate formation. Mutations does not affect the affinity of Ubc2b binding, however, differences in kcat values determined from ternary complex formation versus HsUbc2b transthiolation suggest that binding of the E2 enhances the rate of bound ubiquitin adenylate formation
D576E
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Km-value for ATP is 4fold higher than wild-type value, KM-value for ubiquitin is 1.4fold higher than wild-type value. kcat for ubiquitin adenylate formation is 1200fold lower than wild-type value. kcat for ubiquitin carrier protein E2 transthiolation is 34fold lower than wild-type value; mutation within the MgATP2- binding site, results in dramatically impaired binding affinities for MgATP2-, a shift from ordered to random addition in co-substrate binding, and a significantly reduced rate of ternary complex formation that shifts the rate-limiting step to ubiquitin adenylate formation. Mutations does not affect the affinity of Ubc2b binding, however, differences in kcat values determined from ternary complex formation versus HsUbc2b transthiolation suggest that binding of the E2 enhances the rate of bound ubiquitin adenylate formation
D576N
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Km-value for ATP is 5.2fold higher than wild-type value, KM-value for ubiquitin is 5fold higher than wild-type value. kcat for ubiquitin adenylate formation is 545fold lower than wild-type value. kcat for ubiquitin carrier protein E2 transthiolation is 155fold lower than wild-type value; mutation within the MgATP2- binding site, results in dramatically impaired binding affinities for MgATP2-, a shift from ordered to random addition in co-substrate binding, and a significantly reduced rate of ternary complex formation that shifts the rate-limiting step to ubiquitin adenylate formation. Mutations does not affect the affinity of Ubc2b binding, however, differences in kcat values determined from ternary complex formation versus HsUbc2b transthiolation suggest that binding of the E2 enhances the rate of bound ubiquitin adenylate formation
DELTA946
truncation of the Uba1 carboxyl-terminal beta-grasp domain reduces cognate Ubc2b binding by 31-fold and kcat by 33500-fold; truncation of the Uba3 carboxyl-terminal beta-grasp domain, no effect on cognate Ubc12 thiolester formation
K528A
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Km-value for ATP is 1.6fold higher than wild-type value, KM-value for ubiquitin is 2.9fold higher than wild-type value. kcat for ubiquitin adenylate formation is 400fold lower than wild-type value. kcat for ubiquitin carrier protein E2 transthiolation is 309fold lower than wild-type value; mutation within the MgATP2- binding site, results in dramatically impaired binding affinities for MgATP2-, a shift from ordered to random addition in co-substrate binding, and a significantly reduced rate of ternary complex formation that shifts the rate-limiting step to ubiquitin adenylate formation. Mutations does not affect the affinity of Ubc2b binding, however, differences in kcat values determined from ternary complex formation versus HsUbc2b transthiolation suggest that binding of the E2 enhances the rate of bound ubiquitin adenylate formation
A189T/W714C
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mutant protein is less stable than its wildtype counterpart, and restrictive temperature of 39°C accelerates its degradation
W714C
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the mutant enzyme is less stable than its wild-type counterpart, and restrictive temperature (39°C) accelerates its degradation
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
synthesis