Chemoenzymatic synthesis of K29/K48-branched ubiquitin chains using LbPro* andOaAEP1 enzymes

Branched ubiquitin (Ub) chain modification represents an emerging form of ubiquitination, whose intricate topological architectures enable precise regulation of substrate proteins’ fate. However, the biochemical characterization of branched Ub chains — including deubiquitinase (DUB) selectivity — remains poorly explored, primarily due to challenges in obtaining structurally defined branched Ub chains. Here, we report a chemoenzymatic strategy to synthesize branched Ub chains through the integrated use of two engineered enzymes, LbPro* and OaAEP1. This approach enables precise construction of branched Ub chains via a two-step process: first, generating Ub chain units modified with isopeptide-linked GlyGly via LbPro*–assisted enzymatic assembly; then, ligating these units to other Ub molecules via OaAEP1-mediated transpeptidation. Using this strategy, we successfully prepared K29/K48-branched tri-Ub and tetra-Ub chains. Biochemical hydrolysis assays using these synthetic tri-Ub and tetra-Ub chains demonstrated that the DUB TRABID specifically targets the K29 linkage within these branched Ub chains and exhibits variable cleavage efficiency depending on chain length and topology. Overall, this work expands the chemoenzymatic toolkit for synthesizing branched Ub chains and highlights the importance of using structurally defined branched Ub chains to dissect DUB biochemistry