Illuminating Substrate Preferences of Promiscuous F420H2-Dependent Dehydroamino Acid Reductases with 4-Track mRNA Display

Stereoselective reduction of dehydroamino acids is a common biosynthetic strategy to introduce d-amino acids into peptidic natural products. The reduction, often observed during the biosynthesis of lanthipeptides, is performed by dedicated dehydroamino acid reductases (dhAARs). Enzymes from the three known dhAAR families utilize nicotinamide, flavin, or F₄₂₀H₂ coenzymes as hydride donors, and little is known about the catalysis performed by the latter family proteins. Here, we perform a bioinformatics-guided identification and large-scale in vitro characterization of five F₄₂₀H₂-dependent dhAARs. We construct an mRNA display-based pipeline for ultrahigh throughput substrate specificity profiling of the enzymes. The pipeline relies on a 4-track selection strategy to deliver large quantities of clean data, which were leveraged to build accurate substrate fitness models. Our results identify a remarkably promiscuous enzyme, referred to as MaeJ_C, that is capable of installing d-Ala residues into arbitrary substrates with minimal recognition requirements. We integrate MaeJ_C into a thiopeptide biosynthetic pathway to produce d-amino acids-containing thiopeptides, demonstrating the utility of MaeJ_C for the programmable installation of d-amino acids in ribosomal peptides.