The last step in cocaine biosynthesis is catalyzed by a BAHD acyltransferase.

The esterification of methylecgonine (2-carbomethoxy-3ß-tropine) with benzoic acid is the final step in the biosynthetic pathway leading to the production of cocaine in Erythoxylum coca. Here we report the identification of a member of the BAHD family of plant acyltransferases as cocaine synthase. The enzyme is capable of producing both cocaine and cinnamoylcocaine via the activated benzoyl- or cinnamoyl-Coenzyme A thioesters, respectively. Cocaine synthase activity is highest in young developing leaves, especially in the palisade parenchyma and spongy mesophyll. These data correlate well with the tissue distribution pattern of cocaine as visualized with antibodies. Matrix-assisted laser-desorption ionization mass spectral imaging revealed that cocaine and cinnamoylcocaine are differently distributed on the upper versus lower leaf surfaces. Our findings provide further evidence that tropane alkaloid biosynthesis in the Erythroxylaceae occurs in the above-ground portions of the plant in contrast with the Solanaceae, in which tropane alkaloid biosynthesis occurs in the roots.

The biosynthesis of hydroxycinnamoyl quinate esters and their role in the storage of cocaine in Erythroxylum coca.

Complexation of alkaloids is an important strategy plants utilize to facilitate storage in vacuoles and avoid autotoxicity. Previous studies have implicated hydroxycinnamoyl quinate esters in the complexation of purine alkaloids in Coffea arabica. The goal of this study was to determine if Erythroxylum coca uses similar complexation agents to store abundant tropane alkaloids, such as cocaine and cinnamoyl cocaine. Metabolite analysis of various E. coca organs established a close correlation between levels of coca alkaloids and those of two hydroxycinnamoyl esters of quinic acid, chlorogenic acid and 4-coumaroyl quinate. The BAHD acyltransferase catalyzing the final step in hydroxycinnamoyl quinate biosynthesis was isolated and characterized, and its gene expression found to correlate with tropane alkaloid accumulation. A physical interaction between chlorogenic acid and cocaine was observed and quantified in vitro using UV and NMR spectroscopic methods yielding similar values to those reported for a caffeine chlorogenate complex in C. arabica. These results suggest that storage of cocaine and other coca alkaloids in large quantities in E. coca involves hydroxycinnamoyl quinate esters as complexation partners.