Substrate-Programmed Selectivity in Sequential Transpeptidase Ligation

Enzymes that process two substrates through sequential steps recognize structural motifs on the substrates that define selectivity at each reaction step. However, whether and how selectivity emerges in sequential, multi-substrate reactions remains incompletely understood. Here, we hypothesized that first-substrate binding reshapes the enzyme-substrate intermediate to program second-substrate selectivity. Using a transpeptidase, ancestral sortase variant (AcSE5), as a model to test the hypothesis, we developed a Contact Reporter assay that enables rapid and quantitative profiling of ligation across systematically diversified first-second substrate combinations. This comprehensive analysis revealed that single-residue changes in the first substrate generate distinct selectivity profiles for subsequent nucleophiles. To extend this analysis beyond peptide-based substrates, we further established a fluorescence readout and visualized selectivity across a chemically diverse set of 79 primary amines. Together, our findings reveal how selectivity emerges in sequential, multi-substrate reactions: first-substrate binding biases second-substrate preference via the enzyme–substrate intermediate, offering a generalizable framework for directing ligation outcomes in transpeptidases and related bioconjugation platforms