Strychnine, a poisonous alkaloid from the Strychnos nux-vomica tree, has fascinated chemists for more than 200 years, inspiring them to determine its complex molecular skeleton and to reconstruct the molecule in the lab. But the full biosynthetic pathway that S. nux-vomica uses to build strychnine has been a mystery—until now. Using chemical logic and a study of the messenger RNA S. nux-vomica makes, researchers have discovered the suite of enzymes and chemical steps that lead to strychnine and related compounds (Nature 2022, DOI: 10.1038/s41586-022-04950-4).
Because strychnine is a bioactive molecule, its derivatives could have pharmacologically useful properties without being poisons. Knowing the biosynthetic route could help chemists generate the strychnine scaffold and make such derivatives using synthetic biology, says the Max Planck Institute for Chemical Ecology’s Sarah E. O’Connor, who led the project.
The biosynthetic pathway held some surprises, say O’Connor and Benke Hong, a postdoctoral scholar in O’Connor’s lab and the study’s first author. Hong says he spent several months trying to find enzymes that complete the last step in the biosynthesis—the conversion of prestrychnine to strychnine—with no success. One Friday, he left a sample of prestrychnine on his bench instead of storing it in the freezer. When he examined the sample a few days later, there was some strychnine in the sample with more strychnine appearing over time. This made Hong think that the conversion of prestrychnine to strychnine occurs spontaneously, catalyzed by acidic conditions within the plant rather than with the help of enzymes. Although the researchers can’t rule out that an enzyme performs this step, experiments with radiolabeled strychnine precursors in S. nux-vomica suggest the conversion is occurring spontaneously.
The researchers also studied a plant in the Strychnos family that makes diaboline instead of strychnine. Both biosynthetic pathways involve a common intermediate, so the chemists wondered why they diverged. They discovered that an acetyl transferase enzyme in the plant that makes diaboline has a slightly different amino acid sequence in S. nux-vomica, making it a malonyl transferase enzyme. Instead of putting an acetyl group on the indoline nitrogen to form diaboline, the enzyme adds a malonyl group to that indoline nitrogen to make prestrychnine. “It’s one amino acid that changes the whole pathway from diaboline to strychnine,” O’Connor says.
Yi Tang, an expert in biosynthesis at the University of California, Los Angeles, says the discovery of the malonyl transferase enzyme was unexpected and key to the biosynthetic route. Figuring out the biosynthesis of strychnine, “lays the foundation to engineered biosynthesis of strychnine and related compounds using modern synthetic biology tools,” he says in an email.