Literature summary for 2.3.2.23 extracted from

Schelpe, J.; Monte, D.; Dewitte, F.; Sixma, T.; Rucktooa, P.
Structure of UBE2Z enzyme provides functional insight into specificity in the FAT10 protein conjugation machinery (2016), J. Biol. Chem., 291, 630-639 .

Cloned(Commentary)

Cloned (Comment)	Organism
gene UBE2Z, sequence comparisons, recombinant overexpression of His6-tagged enzyme in Escherichia coli strain BL21(DE3)	Homo sapiens

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant detagged UBE2Z, microcrystals are grown by mixing of 12 mg/ml protein solution with crystallization solution containing 25% w/v PEG 6000 and 0.1 M Tris, pH 8.0, a seed stock is generated from these microcrystals and used in cross-seeding, diffraction quality crystals grow at room temperature in 8-25% w/v PEG 1500, 0.1 M D-malic acid-MES-TRIS buffer at pH 5.0-7.5, X-ray diffraction structure determination and analysis at 2.1 A resolution, molecular replacement using the structure of UBE2D3 (PDB ID 1X23) as the search model for UBE2Z. The determined structure lacks the N-terminal 98 residues of the protein corresponding to the UBE2Z N-terminal extension	Homo sapiens

Crystallization (Comment)

Organism

purified recombinant detagged UBE2Z, microcrystals are grown by mixing of 12 mg/ml protein solution with crystallization solution containing 25% w/v PEG 6000 and 0.1 M Tris, pH 8.0, a seed stock is generated from these microcrystals and used in cross-seeding, diffraction quality crystals grow at room temperature in 8-25% w/v PEG 1500, 0.1 M D-malic acid-MES-TRIS buffer at pH 5.0-7.5, X-ray diffraction structure determination and analysis at 2.1 A resolution, molecular replacement using the structure of UBE2D3 (PDB ID 1X23) as the search model for UBE2Z. The determined structure lacks the N-terminal 98 residues of the protein corresponding to the UBE2Z N-terminal extension

Homo sapiens

Protein Variants

Protein Variants	Comment	Organism
additional information	generation of deletion mutants UBE2ZDELTALB and UBE2ZDELTANter	Homo sapiens

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
additional information	-	additional information	initial rates for UBL transfer from the E1 to the E2 enzymes are determined at different E2 concentrations and are fitted using non-linear regression to the Michaelis-Menten model or to a substrate inhibition effect equation. Data, fitted to the Michaelis-Menten model, shows that wild-type UBE2Z, and mutants UBE2ZLB and UBE2ZNter display similar catalytic rates during ubiquitin loading, Enzyme kinetics for UBL loading on UBE2Z variants, overview	Homo sapiens

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	required in the E2 loading reaction	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	Q9H832	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His6-tagged enzyme UBE2Z from Escherichia coli strain BL21(DE3) by metal affinity chromatography, dialysis, tag cleavage through HRV 3C protease, followed by anion exchange chromatography and dialysis, gel filtration and ultrafiltration	Homo sapiens

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	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	-	?
S-ubiquitinyl-[E1 ubiquitin-activating enzyme]-L-cysteine + [E2 ubiquitin-conjugating enzyme]-L-cysteine	E2 loading reaction assay using ubiquitin D, i.e. FAT10, UniProt ID O15205. UBE2Z is specific for E1-like ubiquitin-activating enzyme UBA6. UBE2Z N-terminal extension and loop LB are essential for selectivity toward UBA6. UBA6 charges UBE2Z with FAT10 less efficiently than with ubiquitin. The C-terminal CYCI peptide in rFAT10 limits transfer Rates onto UBE2Z	Homo sapiens	[E1 ubiquitin-activating enzyme]-L-cysteine + S-ubiquitinyl-[E2 ubiquitin-conjugating enzyme]-L-cysteine	-	?

Subunits

Subunits	Comment	Organism
More	UBE2Z is a 354-residue-long atypical ubiquitin conjugating enzyme comprising about 100-residue long N- and C-terminal extensions on top of the conserved core UBC domain, classifying it as a class IV E2 enzyme. The UBE2Z core domain adopts the characteristic ellipsoid shape of UBC domains but also harbors two extensions termed loops LA (residues 169-173) and LB (residues 194-197) compared with the prototypical class I E2 enzyme UBE2D3. Structural organization of UBE2Z, modeling, overview	Homo sapiens

Synonyms

Synonyms	Comment	Organism
UBE2Z	-	Homo sapiens

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
32	-	assay at	Homo sapiens

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	assay at	Homo sapiens

General Information

General Information	Comment	Organism
evolution	UBE2Z is a 354-residue-long atypical ubiquitin conjugating enzyme comprising about 100-residue long N- and C-terminal extensions on top of the conserved core UBC domain, classifying it as a class IV E2 enzyme	Homo sapiens
additional information	the structure of UBE2Z enzyme provides functional insight into specificity in the FAT10 protein conjugation machinery. UBE2Z is specific for E1-like ubiquitin-activating enzyme UBA6. UBE2Z N-terminal extension and loop LB are essential for selectivity toward UBA6	Homo sapiens
physiological function	UBE2Z is a selective E2 enzyme, functioning in ubiquitination only with E1-like ubiquitin-activating enzyme UBA6	Homo sapiens