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

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
the need for Ub thioester in LACE. The chimeric E1 was subjected to
directed evolution to improve its apparent second-order rate constant
(kcat/KM)
400-fold. We demonstrate the utility of the chimeric E1 by site-specific
transfer of mono- and oligo-Ub to various target proteins in vitro. Additionally, the chimeric E1, Ubc9, Ub, and the
target protein can be coexpressed in Escherichia coli for the facile preparation of monoubiquitylated proteins.