Direct Bioconjugations of Diverse Payloads using an Extraordinarily Donor-Promiscuous Flavin Transferase

Abstract

Site-specific protein conjugation is a widely used tool in chemical biology research and is the foundation of many biotechnological and biomedical applications. A key enabling technology are repurposed post-translational protein modifying enzymes, where variants with broad donor and acceptor substrate promiscuity were developed into general-purpose bioconjugation platforms. However, direct enzymatic attachment of bulky or structurally diverse payloads is often limited by low reaction efficiencies and difficulties in preparing suitable donors. Consequently, conjugation typically relies on two-step strategies in which an enzyme first installs a bioorthogonal handle on the target protein, followed by chemical attachment of the payload. These enzyme-first, chemistry-second approaches limit chemistry to bioorthogonal reactions that leave a molecular footprint. Here, we introduce direct ADDing, an enzymatic bioconjugation platform based on our discovery of the exceptional donor promiscuity of the flavin transferase ApbE. We show that ApbE accepts a remarkably broad range of donor substrates provided they carry an adenosine diphosphate (ADP) anchor. The prevalence of ADP-containing compounds in nature provides access to diverse building blocks and an extensive biochemical toolbox for donor generation. Exploiting these resources, we demonstrate multiple strategies for preparing compatible donors and achieve direct, site-specific enzymatic conjugation of diverse small molecules, peptides, proteins, and synthetic polymers.