Advances in enzymatic reconstitution of ubiquitin chains and substrate ubiquitination

Ubiquitination is a central post-translational modification that regulates processes like protein degradation and signalling. This versatility is derived from ubiquitin (Ub) chains, which are formed via eight distinct linkages. Analysing these complex functions requires the use of Ub chains with precisely defined linkage types and lengths. Although chemical synthesis allows for the preparation of these Ub chains, enzymatic reconstitution offers a more broadly accessible strategy. This review focuses on recent enzymatic advances for the in vitro synthesis of high-purity Ub chains, employing E1-E2-E3 cascades, engineered enzymes and linkage-specific deubiquitinases. Key discussion points include methods to control chain length, prevent unwanted cyclization and generate complex mixed or branched topologies. Furthermore, methods for site-specific substrate ubiquitination are summarized. These robust enzymatic systems are indispensable tools, enabling the reconstruction of complex Ub modifications in vitro and advancing structural and biochemical studies of the Ub code.