Mammalian-like steroidogenesis in plants gives rise to endocrine-mimetic cardenolides

Steroid hormones are central regulators of eukaryotic physiology, yet their signaling pathways are also exploited as key targets in cross-kingdom endocrine interference. Among them, cardenolides are steroid hormones in mammals that target sodium-potassium adenosine triphosphatase (Na⁺/K⁺-ATPase) to modulate ionic homeostasis. Notably, at least 17 plant orders are known to produce cardenolides as potent antiherbivore defenses by mimicking this signaling axis. A prominent example is digoxin, an FDA-approved cardiac drug sourced from foxglove. Despite decades of study, the biochemical basis by which plants evolved this endocrine mimicry remains unclear. Here, we identify S14βH, a noncanonical 2-oxoglutarate/Fe(II)-dependent dioxygenase (2OGD) that catalyzes stereoinverted steroid C14β-hydroxylation through an unprecedented mechanism involving hydrogen atom transfer, substrate reorientation, and opposite-face hydroxyl rebound. An intricate metabolic network comprising enzymatic redox transformations and malonylation, together with spontaneous lactonization, then channels 14β-hydroxylated pregnenolone toward the core cardenolide digitoxigenin. Comparative biochemical and evolutionary analyses further reveal that mammalian-like steroidogenesis is conserved in seed plants and has been independently co-opted for cardenolide biosynthesis across diverse lineages. Our findings expand the mechanistic scope of radical enzymology, illustrate how the malleability of plant metabolism enables repeated endocrine mimicry, and ultimately motivate future investigation into potential hormonal functions of steroids produced via mammalian-like steroidogenesis in plants.