Reprogramming Nonribosomal Peptide Synthetases for “Clickable” Amino Acids

Abstract

AbstractNonribosomal peptide synthetases (NRPSs) are multifunctional enzymes that produce a wide array of bioactive peptides. Here we show that a single tryptophan‐to‐serine mutation in phenylalanine‐specific NRPS adenylation domains enables the efficient activation of non‐natural aromatic amino acids functionalized with azide and alkyne groups. The resulting 105‐fold switch in substrate specificity was achieved without appreciable loss of catalytic efficiency. Moreover, the effective communication of the modified A domains with downstream modules in dipeptide synthetases permitted incorporation of O‐propargyl‐L‐tyrosine into diketopiperazines both in vitro and in vivo, even in the presence of competing phenylalanine. Because azides and alkynes readily undergo bioorthogonal click reactions, reprogramming NRPSs to accept non‐natural amino acids that contain these groups provides a potentially powerful means of isolating, labeling, and modifying biologically active peptides.