Literature summary for 2.8.1.B2 extracted from

Noma, A.; Sakaguchi, Y.; Suzuki, T.
Mechanistic characterization of the sulfur-relay system for eukaryotic 2-thiouridine biogenesis at tRNA wobble positions (2009), Nucleic Acids Res., 37, 1335-1352.

Search for more literature for 2.8.1.B2

Cloned(Commentary)

Cloned (Comment)	Organism
His-tagged Uba4p is recombinantly expressed in Escherichia coli strain Rosetta(DE3). The expression of the recombinant protein is induced by 2% lactose	Saccharomyces cerevisiae

Protein Variants

Protein Variants	Comment	Organism
C225S	when a plasmid harboring UBA4 with a C225S mutation is introduced into the DELTAUBA4 strain, no thiouridine formation of tRNAGlu can be observed, demonstrating that Cys225 is the active-site cysteine residue of UBA4 required for 2-thiouridine formation	Saccharomyces cerevisiae
C397S	approximately half the fraction of tRNAGlu in the DELTAUBA4 strain can be 2-thiolated by the introduction of a plasmid encoding the wild-type UBA4. When pUBA4 C397S is introduced, no 2-thiouridine formation of tRNAGlu occurrs. Cys397 in the rhodanese-like domain (RLD) of UBA4 is critical for 2-thiouridine formation	Saccharomyces cerevisiae

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
Urm1p-terminal-Gly-AMP + Uba4-SSH	Saccharomyces cerevisiae	the substrate Urm1 is an ubiquitin-related modifier involved in protein urmylation. UBA4 is required for cytoplasmic 2-thiouridine formation. Uba4p (a paralog of the ubiquitin-activating enzyme E1) is an enzyme in the ubiquitin-related pathway that activates the C-terminus of Urm1p to form the acyl-adenylated intermediate, then transfers the persulfide sulfur from its C-terminal rhodanese-like domain (RLD) to form thiocarboxylated Urm1p (Urm1p-COSH) by releasing AMP. Urm1p-COSH is a substrate of 2-thiouridine formation catalyzed by Ncs2p and Ncs6p. For protein urmylation, Urm1p-COSH is conjugated with Uba4p, then the putative E3 enzyme transfers Urm1p to target proteins such as Ahp1p	Urm1p-terminal-Gly-COSH + Uba4-SH + AMP	-	?

Organism

Organism	UniProt	Comment	Textmining
Saccharomyces cerevisiae	P38820	-	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
additional information	Uba4p has the potential to act as an acceptor of the persulfide sulfur from Nfs1p, but does not function as an activator for Nfs1p. It is proposed that the persulfide sulfur of Nfs1p is mainly transferred to the rhodanese-like domain 2 (RLD2) Cys259 of Tum1p, and the sulfur is then relayed to the RLD (Cys397) of Uba4p. In addition, direct sulfur transfer from Nfs1p to the RLD (Cys397) of Uba4p also takes place as a minor pathway	Saccharomyces cerevisiae	?	-	?
Urm1p-terminal-Gly-AMP + Uba4-SSH	the substrate Urm1 is an ubiquitin-related modifier involved in protein urmylation. UBA4 is required for cytoplasmic 2-thiouridine formation. Uba4p (a paralog of the ubiquitin-activating enzyme E1) is an enzyme in the ubiquitin-related pathway that activates the C-terminus of Urm1p to form the acyl-adenylated intermediate, then transfers the persulfide sulfur from its C-terminal rhodanese-like domain (RLD) to form thiocarboxylated Urm1p (Urm1p-COSH) by releasing AMP. Urm1p-COSH is a substrate of 2-thiouridine formation catalyzed by Ncs2p and Ncs6p. For protein urmylation, Urm1p-COSH is conjugated with Uba4p, then the putative E3 enzyme transfers Urm1p to target proteins such as Ahp1p	Saccharomyces cerevisiae	Urm1p-terminal-Gly-COSH + Uba4-SH + AMP	-	?
Urm1p-terminal-Gly-AMP + Uba4-SSH	the C-terminal Gly of Urm1p is first activated by Uba4p to synthesize the acyl-adenylated intermediate, then thiocarboxylated by releasing AMP. The rhodanese-like domain (RLD) of UBA4 acts as a persulfide carrier	Saccharomyces cerevisiae	Urm1p-terminal-Gly-COSH + Uba4-SH + AMP	-	?

Synonyms

Synonyms	Comment	Organism
rhodanese-like domain of UBA4	-	Saccharomyces cerevisiae
Uba4p	-	Saccharomyces cerevisiae

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
30	-	assay at	Saccharomyces cerevisiae

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Release 2023.1 (January 2023)