Nett_2023_Nature__

Plants synthesize numerous alkaloids that mimic animal neurotransmitters(1). The diversity of alkaloid structures is achieved through the generation and tailoring of unique carbon scaffolds(2,3), yet many neuroactive alkaloids belong to a scaffold class for which no biosynthetic route or enzyme catalyst is known. By studying highly coordinated, tissue-specific gene expression in plants that produce neuroactive Lycopodium alkaloids(4), we identified an unexpected enzyme class for alkaloid biosynthesis: neofunctionalized alpha-carbonic anhydrases (CAHs). We show that three CAH-like (CAL) proteins are required in the biosynthetic route to a key precursor of the Lycopodium alkaloids by catalysing a stereospecific Mannich-like condensation and subsequent bicyclic scaffold generation. Also, we describe a series of scaffold tailoring steps that generate the optimized acetylcholinesterase inhibition activity of huperzine A(5). Our findings suggest a broader involvement of CAH-like enzymes in specialized metabolism and demonstrate how successive scaffold tailoring can drive potency against a neurological protein target.