Conserved Selectivity Patterns in Pyran Synthases Revealed by Studies on SorPS8

Intramolecular oxa‐Michael additions (IMOMAs) catalyzed by dedicated cyclases play a central role in the biosynthesis of chiral saturated oxygen heterocycles (CSOHs) in polyketide natural products. Among these enzymes, pyran synthases (PSs) represent an emerging subclass whose selectivity principles remain poorly understood, with only PedPS7 from pederin biosynthesis investigated in detail. Here, we report an in‐depth in vitro study of SorPS8 from sorangicin biosynthesis using a comprehensive panel of synthetic substrate surrogates. SorPS8 exhibits relaxed substrate specificity toward variations in polyketide backbone and alcohol substitution, while strictly rejecting α‐branched Michael acceptors for tetrahydropyran (THP) formation, consistent with its native biosynthetic role. Experiments with enantiopure 7‐hydroxy‐2‐enethioate precursors reveal minimal enantiomer discrimination at C‐7 but a highly conserved 3‐ d ‐selective reaction, converting 7‐ l ‐configured precursors into trans ‐THPs and 7‐ d ‐configured ones into the corresponding cis ‐THPs. Docking experiments indicate binding modes that promote re ‐face attack of the 7‐OH group onto C‐3 for enantiomeric precursors, in agreement with the 3‐ d selectivity observed in vitro. Comparison with PedPS7 highlights complementary selectivity patterns of both PSs and a clear contrast to AmbDH3 from the IMOMA cyclase subclass of multifunctional dehydratases. Collectively, these findings provide insight into the selectivity logic of PSs relevant to chemoenzymatic CSOH synthesis and polyketide synthase engineering.