Abstract

Nicotine, tobacco’s addictive and potent insecticidal alkaloid, has shaped human history, agriculture, and the plants that produce it. However, the enzymatic steps and reaction mechanisms involved in nicotine biosynthesis remain elusive. Here, we reveal that the final coupling reaction is stabilized by glycosylation via a uridine diphosphate (UDP)-glycosyltransferase, reduced and activated by an A622, condensed through a stereoselective intermolecular Mannich-like reaction, sequentially oxidized by a berberine bridge enzyme-like (BBL), and finally deglycosylated by a β-glucosidase to yield nicotine. A 5-component metabolon assembles at vacuolar membranes to channel both nicotine biosynthesis and its transport. We reconstituted this metabolon both in vitro and heterologously in vivo . Abrogating any of these components depletes nicotine accumulations. A multidrug and toxic compound extrusion (MATE) transporter is essential for efficiently engineering nicotine production in heterologous plant species, which confers pest resistance. This work completes the nicotine biosynthesis pathway and provides critical insights into the intermolecular Mannich-like reaction, a fundamental mechanism for scaffold formation in many plant alkaloids.