Researchers from Germany have identified the last remaining steps in the biosynthesis of tropane alkaloids from Coca
Tropane alkaloids are a particular class of plant-derived compounds that have been exploited by mankind since the domestication of medicinal plants. The distribution of these alkaloids is scattered amongst the flowering plants and the two most studied families include those from the Solanaceae (tomato, tobacco, potato relatives) and the Erythroxylaceae (coca).
The WHO lists several tropane alkaloids as some of the most important medicines in the modern-day pharmacopeia. However other compounds such as cocaine are more infamous for their narcotic and euphorigenic properties.
Dr John D’ Auria is head of the Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) research group Metabolic Diversity. He said: “It is critical to understand how plants produce these alkaloids in order for mankind to continue to build upon nature and develop new useful medicines.”
The most studied and characterised system for tropane production has historically been within Solanaceae. There are more than ten chemical modification steps necessary to transform the beginning amino acid precursors into the final active alkaloids and all of these steps were identified and characterised in solanaceous plants.
The scattered distribution of tropanes among flowering plants has always hinted that different families may have developed the ability to produce these alkaloids independently from one another. In fact, several steps of tropane biosynthesis were already documented to have evolved independently within members of the Erythroxylaceae.
D’ Auria added: “We have been working on elucidating the coca-derived tropane pathway for the last 15 years and we have been successful in working on several key steps in the biosynthesis of cocaine and other related tropanes in coca.
“The idea that coca would share similar enzymes and genes with their distant solanaceous relatives was incorrect. While the final structure of tropanes is similar, the pathway leading to these alkaloids is different.”
In order to discover the last remaining steps of the pathway in coca, D’ Auria collaborated with the lab of Dr Christina Smolke from Stanford University. The Smolke group are experts at manipulating yeast and micro-organisms to produce important medicinal compounds via synthetic biology methods.
D’ Auria advised: “With their assistance, we used the multiplicative power of gene manipulation in yeast to test many different gene candidates for the missing steps in the coca pathway. In essence, at every unknown step, we designed and tested multiple candidate sequences.”
These candidate sequences originated from transcriptome studies performed D’ Auria’s group as well as the group of Dr Lyndel Meinhardt from the USDA in Beltsville, Maryland (USA).
D’ Auria continued: “Using this powerful gene discovery platform, we successfully identified all the remaining ‘missing steps’ for tropane biosynthesis in coca. This represents the culmination of more than ten graduate student projects in my group and 15 years of my research.”
mix and match
The most significant portions of the findings now confirm that tropane biosynthesis has independently evolved at least twice during the evolution of flowering plants.
“This is important because we also show in our study that you can mix and match the Solanaceae and Erythroxylaceae genes and produce tropanes,” the IPK researcher said.
In layman’s terms, the research provides multiple tools for synthetic biologists to begin designing the tropane alkaloid pathway in organisms that have never produced them before, and with the ability to use different enzymes for similar steps, it is possible to optimise or modify those steps for specific chemical outcomes.
First author Benjamin Chavez, a PhD student in the D’Auria laboratory, added: “In addition, we also show that the beginning portion of the pathway in coca proceeds by an interesting ‘detour’ or alternate route that doesn’t exist in solanaceous species.
“This provides insights into how plant metabolism can find solutions to biochemical challenges. Namely, we can understand the interplay between early precursors and their bottlenecks.”
Lastly, the researchers discovered a specific enzyme that is responsible for the so-called ‘carbomethoxy group’ present exclusively in coca alkaloids. Solanaceous species do not have this modification. The carbomethoxy group is partially responsible for the euphorigenic properties of cocaine.
Image: A flower from the coca plant, Erythroxylum coca Lam. Benjamin Chavez and colleagues used a yeast-based synthetic biology platform to elucidate the last remaining enzymatic steps involved in tropane alkaloid formation in Erythroxylum coca. Their findings revealed a nearly complete independent origin has evolved when compared to the solanaceous pathway. Danny Kessler.