Chemists in China have discovered two missing steps in the biosynthesis of cocaine in the coca bush (Erythroxylum novogranatense). They corrected the previously proposed erroneous biosynthetic pathway and found the correct one, which was fully reproduced in Bentham's tobacco (Nicotiana benthamiana). The study was published in the Journal of the American Chemical Society.
Cocaine is a natural alkaloid synthesized by plants of the Erythroxylaceae family common in South America. People have been using their leaves since very early times. For example, in 2010, scientists found that already about 8,000 years ago, the inhabitants of the Nanchok Valley in Peru not only chewed coca leaves, but also knew how to extract mixtures of natural alkaloids from them. Europeans discovered cocaine in the 19th century, using it as an anesthetic and even adding it to some drinks. But soon it became clear that cocaine is highly addictive, and its use was significantly reduced.
Most alkaloids from coca leaves have a similar structure, based on the bicyclic amine tropane. Cocaine molecules differ from tropane by having two additional ester groups linked to a bicyclic backbone. And although chemists are able to synthesize cocaine and related alkaloids, they still have not figured out the natural way of its synthesis.
But chemists led by Huang Sheng-Xiong of the Chinese Academy of Sciences' Kunming Institute of Botany have finally figured out how cocaine biosynthesizes. From previous experiments with isotope labels, scientists knew that cocaine and its related alkaloid hyoscyamine are derived from a common precursor, 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoic acid (MPOA). According to most scientists, this acid was converted to cocaine in two stages: first, methylation of the acid group, then, oxidative cyclization. Therefore, Chinese chemists decided to start their research by looking for a methyltransferase capable of converting MPOA to its methyl ester.
First, scientists analyzed the transcriptome of the coca bush (Erythroxylum novogranatense) and found in it the genes responsible for the synthesis of 23 different methyltransferases, hypothetically capable of methylating MPOA. Scientists expressed the most suitable of them in tobacco leaves, but they failed to detect significant amounts of MPOA methyl ester in them. At the same time, scientists were looking for enzymes capable of catalyzing oxidative cyclization, but these searches also led to failure.
Then the chemists suggested that the dominant hypothesis about the biosynthesis of cocaine is incorrect, and the MPOA acid first forms a cycle, and only then is methylated. To support their hypothesis, the researchers expressed different methyltransferase-oxidase pairs in tobacco leaves, and found that the combination of EnMT4 methyltransferase and EnCYP81AN15 oxidase resulted in the formation of large amounts of cocaine and the by-product tropinone. This is how chemists realized that MPOA, under the action of EnCYP81AN15 oxidase, is converted into the cyclic alkaloid ecgonine, which is then methylated in the presence of EnMT4 to form cocaine. At the same time, ecgonine itself is unstable and is easily destroyed with the formation of side tropinone - this was confirmed by experiments with synthetically obtained ecgonine.
As a result, scientists have found the enzymes responsible for two missing steps in the biosynthesis of cocaine. They expressed these and several other essential enzymes in tobacco leaves, resulting in a tobacco that produces up to 400 nanograms of cocaine per milligram of dried leaves.