Widodo, W. S.; Fürst, M. J. L. J.

Site-specific attachment of biorthogonal handles to proteins is an essential tool in chemical biology research and diverse applications including imaging and protein immobilization, as well as for the development of next-generation therapeutics such as antibody drug-conjugates. Among the available methods, enzymatic post-translational modification of short protein tags offers precision, stability, and modularity. However, broader application is often limited by complex substrate syntheses, the requirement of long or rigid recognition tags, and limited reaction efficiencies. Here, we present ADDing, a straightforward enzymatic method for functionalizing proteins with click chemistry handles using the flavin transferase ApbE. We discovered that, given a dedicated adenine diphosphate derivative (ADD) substrate, the enzyme attaches a phosphoribosyl moiety bearing bioorthogonal handles to proteins featuring a DxxxGAT amino acid motif. As the substrates can easily be enzymatically synthesized from NAD and inexpensive precursors, ADDing click handles can be performed in a streamlined, one-pot workflow combining substrate synthesis and protein conjugation. ADDing allows rapid, high-yield functionalization of proteins featuring the recognition tag at either terminus or internal loops and is compatible with copper and copper free azide-alkyne cycloaddition reactions. To demonstrate its broad applicability, we performed a wide variety of protein functionalizations, including fluorescent labeling, protein-protein, protein-DNA conjugation, and protein immobilization. This versatile technology thus holds great potential for chemical biology and the production of biological therapeutics.