Engineered reactivity of a bacterial E1-like enzyme enables ATP-driven modification of protein and peptide C termini.

In biological systems, adenosine triphosphate (ATP) provides an energetic driving force for peptide bond formation, but protein chemists lack tools that emulate this strategy. Here we develop an ATP-driven platform for C-terminal activation and peptide ligation based on MccB, a bacterial ancestor of ubiquitin-activating (E1) enzymes. We show that MccB can act on non-native substrates to generate an O-AMPylated electrophile that reacts with exogenous nucleophiles to form diverse C-terminal functional groups including thioesters, a versatile class of biological intermediates that have been exploited for protein C-terminal bioconjugation. By mining the natural diversity of the MccB family, we identify both epitope-specific and more promiscuous MccBs. We show that epitope-specific MccB activity can be directed toward specific proteins of interest to enable high-yield, ATP-driven protein bioconjugation, and promiscuous MccB activity can be deployed for the synthesis of peptide thioester substrates for bioconjugation. Our method mimics the chemical logic of biological peptide bond synthesis for high-yield in vitro manipulation of protein structure with molecular precision.