Final step in the biosynthesis of iridoids elucidated

Iridoids are a widespread and evolutionarily ancient class of plant secondary metabolites belonging to the terpenes. They occur in thousands of plant species and play an important role in defense and other interactions between plants and their environment. Iridoids are also found in foods such as olives and blueberries, and are believed to have anti-inflammatory properties. They are also essential precursors for many medically important compounds, including the cancer drug vinblastine and the ipecacuanha alkaloids found in the medicinal plants, sage-leaved alangium and ipecac root (see press release Two plant species invent the same chemically complex and medically interesting substance, June 3, 2025). Despite their importance, the biosynthetic pathway had not yet been fully elucidated. The crucial step of cyclisation to nepetalactol — the basic structure of all iridoids — was still missing.

Sarah O'Connor, head of the Natural Product Biosynthesis Department at the Max Planck Institute for Chemical Ecology in Jena, has been researching this biosynthetic pathway for over 15 years. 'Enzymes that can form the iridoid skeleton have been known for a long time. These iridoid synthases produce small amounts of nepetalactol and large amounts of by-products simultaneously. We therefore long suspected that the crucial cyclisation reaction could occur spontaneously, without the help of another enzyme. However, later experiments, including those on catnip (see press release How mint became catmint, May 14, 2020), provided evidence that cyclisation is catalyzed by an enzyme. Nevertheless, we had no idea what such an enzyme might look like,' says Sarah O'Connor, explaining the starting point of the research. As it was not possible to narrow the search down to specific enzyme classes, hundreds of possible candidates would normally have had to be tested for activity. However, thanks to a collaboration with Robin Buell from the University of Georgia (USA), a new dataset became available that proved crucial in narrowing down the number of candidates.

Generated by Robin Buell's laboratory using state-of-the-art methods, this dataset included gene expression in individual ipecac cells, rather than in different tissues as in a previously compiled dataset. Comparing the two datasets revealed that only a very small number of unknown genes correlated with the expression of the known iridoid biosynthesis genes in both datasets. This small number enabled rapid testing for activity, which was carried out by master's student Chloée Tymen. For one of these unknown genes, the researchers were able to demonstrate that the cyclisation reaction is catalyzed when it is expressed in plants or bacteria. This confirmed that this gene encodes the long-sought cyclase. Comparing the amino acid sequence of the cyclase with sequences from thousands of plant species in public databases revealed that the enzyme occurs precisely in plant species that form iridoids. 'We were able to show that the reaction we investigated is indeed catalyzed by the enzyme and that the expected substance, nepetalactol, is formed.' However, to our surprise, the enzyme we found belongs to a completely unexpected class of enzymes. This class of enzymes is known for catalyzing a completely different reaction," explains Maite Colinas, group leader in Sarah O'Connor's department and first author of the study.

The mechanistic process of cyclisation is still unclear. There are different ways in which a cyclization can occur chemically, and it is not yet known through which of the possible chemical reaction mechanisms the discovered cyclisation proceeds. It is also unclear how the cyclisation function evolutionary evolved from a completely different enzyme reaction. These questions are the subject of further investigation.

The discovery of the cyclase important for iridoid formation shows once again that enzymes can catalyze completely unexpected reactions that cannot be predicted by bioinformatic methods. This enzyme paves the way for future biotechnological production of nepetalactol and its derivatives, such as the anti-cancer drugs vinblastine and vincristine, in yeast, fungi, or other plant species.