Illuminating Substrate\nPreferences of Promiscuous\nF<sub>420</sub>H<sub>2</sub>‑Dependent Dehydroamino Acid Reductases\nwith 4‑Track mRNA Display

Stereoselective reduction of dehydroamino acids is a\ncommon biosynthetic\nstrategy to introduce d-amino acids into peptidic natural\nproducts. The reduction, often observed during the biosynthesis of\nlanthipeptides, is performed by dedicated dehydroamino acid reductases\n(dhAARs). Enzymes from the three known dhAAR families utilize nicotinamide,\nflavin, or F<sub>420</sub>H<sub>2</sub> coenzymes as hydride donors,\nand little is known about the catalysis performed by the latter family\nproteins. Here, we perform a bioinformatics-guided identification\nand large-scale in vitro characterization of five F<sub>420</sub>H<sub>2</sub>-dependent dhAARs. We construct an mRNA display-based pipeline\nfor ultrahigh throughput substrate specificity profiling of the enzymes.\nThe pipeline relies on a 4-track selection strategy to deliver large\nquantities of clean data, which were leveraged to build accurate substrate\nfitness models. Our results identify a remarkably promiscuous enzyme,\nreferred to as MaeJ<sub>C</sub>, that is capable of installing d-Ala residues into arbitrary substrates with minimal recognition\nrequirements. We integrate MaeJ<sub>C</sub> into a thiopeptide biosynthetic\npathway to produce d-amino acids-containing thiopeptides,\ndemonstrating the utility of MaeJ<sub>C</sub> for the programmable\ninstallation of d-amino acids in ribosomal peptides.