Iterative acylation on mature lasso peptides by widespread acetyltransferases for lipolasso production

The biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs) leverages iterative catalysis to enhance structural and biological diversity. Traditionally, iterative enzymes install multiple post-translational modifications (PTMs) on linear peptides, rather than mature RiPPs with intricate three-dimensional structures, which would require complex changes in substrate binding poses. Here, we present a prolific class of GCN5-related N-acetyltransferases (GNATs) that iteratively and consecutively acylate two Lys residues within the loop and ring motifs of lasso peptides, diverging from the typical iterative modification of linear peptides—an unprecedented function for PTM enzymes. Utilizing high-resolution cryo-electron microscopy and enzymatic reconstitution, we mapped the lasso peptide binding pocket of IatT and pinpointed key residues involved in demarcating the two distinct acetylation steps. Structure-based engineering of IatT’s acetyl group recognition site expanded the cavity to accommodate longer-chain acyl groups, leading to the creation of lipolasso peptides, a novel class of ribosomal lipopeptide. This engineering strategy can be applied to any RiPP BGC encoding GNAT, facilitating the efficient diversification of rare ribosomal lipopeptides.