Facile One-Step Assembly of Bona Fide SUMO Conjugates by Chemoenzymatic Ligation

The post-translational conjugation of the small ubiquitin-like modifiers (SUMOs) to target proteins occurs through a complex machinery that involves sequential action of at least three enzymes. SUMOylation performs crucial regulatory functions in several cellular processes. The availability of well-defined SUMO conjugates is necessary for untangling the mechanism of SUMOylation. However, assembly of homogeneous SUMO conjugates represents a challenge because of the multi-step synthesis involved and the unwieldiness of the reconstituted biosynthetic systems. Here we describe a simple one-step chemoenzymatic strategy for conjugating engineered SUMO (eSUMO) proteins to a prefabricated isopeptide-linked SUMO target peptide. Notably, the eSUMOs were efficiently recognized by the enzymes of the SUMOylation machinery and the SUMO conjugates served as bona fide substrates for DeSUMOylating enzymes.