Family of β-Propeller Diels–Alderases Directs cis -Decalin Formation in Polyketide Biosynthesis

Abstract Decalin-forming Diels–Alderases (pericyclases) exemplify how Nature repeatedly recruits unrelated protein folds to control [4 + 2] cycloadditions, yet exo-selective intramolecular Diels–Alder (IMDA) reactions that yield cis-fused decalins remain rare and poorly understood. Here, we uncover a previously unrecognized bacterial pericyclase family that enforces an exo-IMDA trajectory in polyketide assembly. Using alchivemycin A, a macrocyclic antibacterial polyketide whose scaffold is defined by a highly functionalized cis-decalin core, we identify AvmG as the dedicated decalin-forming enzyme. Disruption of avmG diverts pathway flux to trans-decalin shunt isomers consistent with spontaneous, endo-biased cyclization that cannot be processed by the downstream macrocyclase, whereas AvmG enables formation of the authentic cis-decalin intermediate. AvmG is unrelated to known [4 + 2] cyclases and adopts an integrin α N-terminal domain-like β-propeller architecture, revealing an unexpected scaffold for pericyclic stereocontrol. Genome mining shows that AvmG-like proteins are widespread and enriched in actinomycete polyketide gene clusters; characterization of the lydicamycin homolog LymR establishes conserved exo-IMDA activity in a distinct pathway. Structural and mutational analyses implicate a loop-defined catalytic cavity in steering the kinetically demanding cis-decalin outcome. Together, these findings expand the fold and mechanistic diversity of Diels–Alderases, define an exo-selective β-propeller pericyclase superfamily, and provide a new handle for mining and bioengineering cis-decalin biosynthetic pathways.