Substrate Length Controls Atropopeptide Complexity and Triggers Excision-Driven Scaffold Hop to Diketopiperazine Alkaloids

Atropopeptides are a family of ribosomally synthesized and posttranslationally modified peptides (RiPPs) that share conserved cross-link-forming amino acids in their core peptides, yet adopt architectures ranging from simple monocyclic to complex polycyclic structures with intriguing three-dimensional shapes. These complex architectures arise from multiple C-C and C-N cross-links that are installed by a single cytochrome P450 enzyme, but the factors governing these divergent cross-linking patterns and resulting structural complexities have remained elusive. Recent studies on RiPP biosynthesis have revealed a form of substrate-controlled catalysis, in which the precursor sequence, rather than the modifying enzyme, dictates the reaction course. Here, we describe a distinct form of substrate-controlled catalysis in atropopeptide biosynthesis, demonstrating that core peptide length, rather than sequence, determines the cross-linking pattern installed by an atropopeptide-forming cytochrome P450. We further uncover an unusual proteolytic excision of an internal amino acid in mature atropopeptides, generating peptides that spontaneously convert into pyrroloindoline-containing diketopiperazines (DKPs) and thereby effecting a nonenzymatic scaffold-hop from atropopeptides to DKP-derived alkaloids, for which we provide mechanistic insight. Both the atropopeptide and the corresponding DKP exhibit antiviral activity, and their structural diversity can be further expanded through precursor engineering. Our work reveals substrate length as a key determinant of atropopeptide complexity, provides insights into the catalysis of a multicross-link-forming P450, and establishes an unprecedented route connecting atropopeptides to pyrroloindoline DKP alkaloids.