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Literature summary for 2.3.2.23 extracted from
- Mechanism of lysine 48 selectivity during polyubiquitin chain formation by the Ube2R1/2 ubiquitin-conjugating enzyme (2016), Mol. Cell. Biol., 36, 1720-1732 .
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| gene UBE2R1 or CDC34, recombinant expression of GST- or His6-tagged enzyme in Escherichia coli strain Rosetta (DE3) | Homo sapiens |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| D102K | site-directed mutagenesis, analysis of ubiquitin specificity and reaction kinetics compared to wild-type | Homo sapiens |
| D103K | site-directed mutagenesis, analysis of ubiquitin specificity and reaction kinetics compared to wild-type | Homo sapiens |
| D143A | site-directed mutagenesis, analysis of ubiquitin specificity and reaction kinetics compared to wild-type | Homo sapiens |
| D143K | site-directed mutagenesis, analysis of ubiquitin specificity and reaction kinetics compared to wild-type | Homo sapiens |
| D143R | site-directed mutagenesis, analysis of ubiquitin specificity and reaction kinetics compared to wild-type | Homo sapiens |
| D91K | site-directed mutagenesis, analysis of ubiquitin specificity and reaction kinetics compared to wild-type | Homo sapiens |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| additional information | - |
additional information | Michaelis constants for wild-type and mutant E2-E3 pairs, E2 is Ube2R2 and E3 is Rbx1, overview | Homo sapiens | |
| 1 | - |
S-ubiquitinyl-[E1 ubiquitin-activating enzyme]-L-cysteine | about, pH 7.5, temperature not specified in the publication | Homo sapiens |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| S-ubiquitinyl-[E1 ubiquitin-activating enzyme]-L-cysteine + [E2 ubiquitin-conjugating enzyme]-L-cysteine | Homo sapiens | - |
[E1 ubiquitin-activating enzyme]-L-cysteine + S-ubiquitinyl-[E2 ubiquitin-conjugating enzyme]-L-cysteine | - |
? |  |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Homo sapiens | P49427 | - |
- |
| Homo sapiens | Q712K3 | - |
- |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| recombinant GST- or His6-tagged enzyme UBE2R1 from Escherichia coli strain Rosetta (DE3) by glutathione or nickel affinity chromatography, respectively, followed by tag cleavage through TEV protease, and gel filtration | Homo sapiens |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| additional information | diubiquitin synthesis by enzyme UBE2R2 with 32P-labeled K48R donor ubiquitin and acceptor ubiquitin, which contains an additional Asp residue at its C-terminus (referred to as D77 ubiquitin, which cannot be thioesterified to Ube2R2), analysis of activities of wild-type Ube2R2 and mutants, D143K, D143R, D143A, and D91K, with wild-type ubiquitin and mutant ubiquitins, R54D, R54A, I44A, I44D, and D58R. The mutations reduce the activity, the combination of D143K enzyme with D58R ubiquitin is inactive, the mutation of Arg54 to an Asp residue in acceptor ubiquitin leads to a 52fold reduction in Ube2R2 activity compared with wild-type ubiquitin results. The Ube2R1/2 acidic loop participates in Lys48-specific polyubiquitin chain formation by binding to Skp1-cullin-Fbox (SCF), kinetics | Homo sapiens | ? | - |
- |
|
| additional information | the Ube2R1/2 acidic loop participates in Lys48-specific polyubiquitin chain formation by binding to Skp1-cullin-Fbox (SCF), kinetics | Homo sapiens | ? | - |
- |
|
| S-ubiquitinyl-[E1 ubiquitin-activating enzyme]-L-cysteine + [E2 ubiquitin-conjugating enzyme]-L-cysteine | - |
Homo sapiens | [E1 ubiquitin-activating enzyme]-L-cysteine + S-ubiquitinyl-[E2 ubiquitin-conjugating enzyme]-L-cysteine | - |
? | |
| S-ubiquitinyl-[E1 ubiquitin-activating enzyme]-L-cysteine + [E2 ubiquitin-conjugating enzyme]-L-cysteine | acceptor ubiquitin residue Arg54 participated in Ube2R1-catalyzed Lys 48 polyubiquitin chain formation. Significance of the Asp143 Ube2R1/2-Arg54 acceptor ubiquitin ion pair | Homo sapiens | [E1 ubiquitin-activating enzyme]-L-cysteine + S-ubiquitinyl-[E2 ubiquitin-conjugating enzyme]-L-cysteine | - |
? | |
| S-ubiquitinyl-[E1 ubiquitin-activating enzyme]-L-cysteine + [E2 ubiquitin-conjugating enzyme]-L-cysteine | acceptor ubiquitin residue Arg54 participates in Ube2R1-catalyzed Lys48 polyubiquitin chain formation. Significance of the Asp143 Ube2R1/2-Arg54 acceptor ubiquitin ion pair | Homo sapiens | [E1 ubiquitin-activating enzyme]-L-cysteine + S-ubiquitinyl-[E2 ubiquitin-conjugating enzyme]-L-cysteine | - |
? | |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| UBE2R1 | - |
Homo sapiens |
| UBE2R2 | - |
Homo sapiens |
| ubiquitin-conjugating enzyme | - |
Homo sapiens |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 7.5 | - |
assay at | Homo sapiens |
General Information
| General Information | Comment | Organism |
|---|---|---|
| additional information | construction of initial Ube2R1-Rbx1 models, Rbx1 (PDB ID 2LGV) and Ube2R1 (PDB ID 4MDK) are aligned to the respective components from the Ube2G2/Rnf45 cocrystal structure (PDB ID 2LXP) and energy is minimized with Rosetta algorithms relax and fixbb. Next, an initial conformation for the acidic loop (residues 97 to 115) is built onto the top-scoring Ube2R1-Rbx1 model using the CCD algorithm with fragments derived from the Ube2R1 amino acid sequence. This model of the Ube2R1-Rbx1 complex is used to create initial models of the Ube2R1-donor ubiquitin/acceptor ubiquitin-Rbx1 complex using the UBQ_E2_thioester protocol, which allows the user to sequentially model the orientation of the thioesterified donor ubiquitin and the approach of an acceptor ubiquitin and to perform standard loop modeling on the acidic loop. The acceptor ubiquitin is based on an apo structure (PDB ID 1UBQ). To generate models consistent with the crystallized orientation of donor ubiquitin, a constraint was implemented between Leu129 on Ube2R1 and both Ile44 and Val70 on the donor ubiquitin. Using this procedure, 4000 theoretical models of Ube2R1-donor ubiquitin/acceptor ubiquitin-Rbx1 are generated yielding 286 low-scoring models. Refining the acceptor ubiquitin and acidic loop. Ube2R1 structural modeling, overview. Molecular modeling of the Ube2R1-donor ubiquitin/acceptor ubiquitin-Rbx1 complex results in 14 distinct clusters of the Ube2R1-acceptor ubiquitin conformation | Homo sapiens |
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