Lysine Side-Chain-to-Backbone Macroamidine Formation Reveals Activation-First YcaO Catalysis

Abstract YcaO enzymes use ATP to modify chemically inert peptide-backbone amides, but the order of catalytic steps has remained unresolved. Here, we characterize a minimal Lysobacter ribosomally synthesized and post-translationally modified peptide (RiPP) pathway in which the stand-alone YcaO LmaB sequentially forms two lysine side-chain-to-backbone macroamidine linkages in the precursor peptide LmaA, revealing a previously unrecognized mode of YcaO-catalyzed peptide cyclization. Bioinformatic analyses place this pathway within a broader class of macroamidine-containing RiPPs, collectively termed macroamitides, which also includes the antibiotic bottromycins. High-resolution LmaB–LmaA structures directly resolve an O-phosphorylated peptide-backbone intermediate coordinated by ADP and three Mg2+ ions and capture the first macroamidine together with O-phosphorylation at the second reaction site. These snapshots reveal iterative substrate repositioning within a single active site. A structure of LmaB H75A retains the O-phosphorylated intermediate but traps the intact lysine nucleophile in a nonproductive conformation, identifying H75 as the general base that activates and positions the lysine ε-amino group for attack. Alanine scanning further reveals extensive tolerance of both ring-forming sequences. Together, these data establish that backbone O-phosphorylation precedes nucleophilic attack, revise the prevailing YcaO mechanism, and provide a model for engineering peptide-backbone chemistry during macroamidine formation.