Nicotine biosynthesis completed by cryptic activating glucosylation

ABSTRACT

Abstract Nicotine is a neuroactive alkaloid produced by tobacco ( Nicotiana tabacum ) as a defense against herbivory, and an addictive stimulant that has been used by humans for millennia. Despite its significance, the core steps of its biosynthesis have remained elusive. Here, we demonstrate in vitro reconstruction of nicotine synthase, a four-enzyme stereoselective biocatalytic cascade that forms ( S )-nicotine from nicotinic acid and N -methylpyrrolinium. This cascade includes two glucose-processing enzymes that participate in a cryptic activating glucosylation step. We also reconstruct this pathway in planta and present high resolution X-ray structures of the key oxidoreductases A622 and BBL bound to their substrate and product, respectively. This work establishes the complete biosynthetic pathway to nicotine, providing new gene targets for controlling alkaloid production in Nicotiana and unlocking enzymatic routes to pyridine alkaloids.