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 IatTs 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. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=95 SRC="FIGDIR/small/630886v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1d88af9org.highwire.dtl.DTLVardef@18c1298org.highwire.dtl.DTLVardef@118b9f3org.highwire.dtl.DTLVardef@580a50_HPS_FORMAT_FIGEXP M_FIG C_FIG