Biosynthesis of YM-216391 Unveils a Bifunctional α-Ketoglutarate-Dependent Enzyme Catalyzes Hydroxylation and Epimerization in RiPPs.

Nonheme iron/α-ketoglutarate-dependent dioxygenases (Fe/αKGDs) catalyze diverse oxidative transformations, including hydroxylation, desaturation, and ring expansion. Although these reactions are well established, Fe/αKGD-catalyzed epimerization remains rare and has not been characterized in ribosomally synthesized and post-translationally modified peptides (RiPPs). Here, we report the RiPP-modifying enzyme YmI, a bifunctional Fe/αKGD that catalyzes Cβ-hydroxylation of Phe19 and Cα-epimerization of Ile20 on the precursor peptide YmA toward the formation of YM-216391. The hydroxylation generates (2S,3R)-β-hydroxyphenylalanine, whose l-threonine-like stereochemistry facilitates subsequent formation of an unusual 5-phenyloxazole moiety. Substrate profiling analyses reveal leader peptide dependence for catalytic activity. Structural and docking studies provide insights into the substrate binding and active site architecture. Deuterium labeling demonstrates that YmI catalyzes Ile epimerization by directly abstracting the Cα-hydrogen, generating a Cα radical that is subsequently reprotonated by a nonexchangeable hydrogen source within the enzyme. These results unveil an unprecedented mechanism and novel bifunctionality, markedly expanding the reaction landscape for αKG-dependent enzymes in RiPP biosynthesis.