Biosynthetic Origin of the Methoxy Group in Quinine and Related Alkaloids.

Quinine is a historically important natural product containing a methoxy group that has been assumed to be incorporated at a late pathway stage. Here we show that the methoxy group in quinine and related alkaloids is introduced onto the starting substrate tryptamine. Feeding studies definitively show that 5-methoxytryptamine is utilized as a quinine biosynthetic intermediate in planta. We discover the biosynthetic genes that encode the responsible oxidase and methyltransferase, and we use these genes to reconstitute the early steps of the alkaloid biosynthetic pathway in Nicotiana benthamiana to produce a mixture of methoxylated and non-methoxylated alkaloid intermediates. Importantly, we show that the co-occurrence of both tryptamine and 5-methoxytryptamine substrates, along with the substrate promiscuity of downstream pathway enzymes, enable parallel formation of both methoxylated and non-methoxylated alkaloids.