Enzymatic Stereoselective Nucleophilic Cyclization Governs Atypical Spirotetronate Assembly in Lucensimycin A Biosynthesis

Lucensimycin A is a structurally unique spirotetronate polyketide featuring a rare spiro[tetronate-hydrophenanthrene] tetracyclic core, distinct from the classical spiro[tetronate-cyclohexene] scaffolds formed via intramolecular Diels-Alder (IMDA) cyclizations. Here, we identified and characterized the luc biosynthetic gene cluster from Streptomyces fagopyri NAX0062, revealing a divergent biosynthetic logic. The pathway begins with type I PKS assembly of a linear polyketide, followed by tetronate ring formation by a canonical tetronate cassette. A flavin-dependent Diels-Alderase (LucM) then catalyzes an IMDA reaction to form a decalin intermediate. Unusually, the Diels-Alderase homologue LucK catalyzes a stereoselective intramolecular nucleophilic cyclization─rather than a pericyclic reaction─to generate the spiro[tetronate-hydrophenanthrene] core, following acetylation by LucN. Oxidative cleavage of a terminal alkene (by LucO3) completes the pathway. Structural and mutational analysis of LucK revealed that Glu16 and Glu85 function as general acid/base catalysts to drive the nucleophilic cyclization reaction, highlighting LucK as a mechanistically distinct cyclase. This work uncovers a previously unrecognized enzymatic strategy for spirocyclic construction and expands the catalytic repertoire of β-barrel enzymes in polyketide biosynthesis.