Site-Specific Protein Ubiquitylation Using an Engineered, Chimeric E1 Activating Enzyme and E2 SUMO Conjugating Enzyme Ubc9.

ABSTRACT: Ubiquitylation-the attachment of ubiquitin (Ub) to proteins in eukaryotic cells-involves a vast number of enzymes from three different classes, resulting in heterogeneous attachment sites and ubiquitin chains. Recently, we introduced lysine acylation using conjugating enzymes (LACE) in which ubiquitin or peptide thioester is site-specifically transferred to a short peptide tag by the SUMO E2 conjugating enzyme Ubc9. This process, however, suffers from slow kinetics-due to a rate-limiting thioester loading step-and the requirement for thioesters restricts its use to in vitro reactions. To overcome these challenges, we devised a chimeric E1 containing the Ub fold domain of the SUMO E1 and the remaining domains of the Ub E1, which activates and loads native Ub onto Ubc9 and obviates th

SUBMITTER: Akimoto G 

PROVIDER: S-EPMC8883482 | biostudies-literature | 2022 Feb

REPOSITORIES: biostudies-literature