PaaA is a RiPP enzyme that catalyzes the transformation of two glutamic acid residues within a substrate peptide into the bicyclic core of Pantocin A. Here, for the first time, we use mRNA display techniques to understand RiPP enzyme-substrate interactions to illuminate PaaA substrate recognition. Additionally, our data revealed insights into the enzymatic timing of glutamic acid modification. The technique developed is quite sensitive and a significant advancement over current RiPP studies and opens the door to enzyme modified mRNA display libraries for natural product-like inhibitor pans.