Exploring the Biosynthesis of Cardenolides in Plant Species

Researchers at the Max Planck Institute for Chemical Ecology in Jena are delving into the previously elusive biosynthetic route responsible for the creation of cardenolides in plants.

In a recently published study in the journal Nature Plants, they unveil two enzymes from the CYP87A family as pivotal catalysts that facilitate the production of pregnenolone, the precursor for plant steroid biosynthesis, in distinct plant families. This discovery holds the potential to pave the way for the development of cost-effective and eco-friendly platforms for generating top-quality steroid compounds for medical purposes.

Plants are prolific producers of an impressive array of metabolites, including medically valuable steroids. Among these compounds, cardenolides stand out. As far back as 1785, the British physician William Withering (1741–1799) documented the red foxglove’s medical applications in his book “An Account of the Foxglove,” revealing its effectiveness in treating water retention by increasing urine flow. What he didn’t realize at the time was that the active components in foxglove leaves directly influenced the heart.

Since the latter half of the 19th century, cardenolides, cardiac glycosides derived from plants, have been employed to manage heart failure and arrhythmia due to their cardiac muscle-affecting properties. Moreover, in recent years, cardenolides have shown considerable success in treating various cancers. Despite their pivotal role in human medicine, the biosynthetic pathways of these steroid molecules in plants have largely remained enigmatic. The primary objective of the research was to uncover how plants synthesize these highly intricate compounds from their theoretically simple precursors, as elucidated by first author Maritta Kunert.

The research team didn’t limit their investigations to just one plant species; they also explored the rubber tree Calotropis procera, despite its belonging to a different plant family. Surprisingly, both plants produced significant quantities of cardenolides.

Since the species under study lacked sequenced genomes and well-documented gene functions, the project initially appeared as a “black box” for the researchers, as they had no established datasets or conventional methods to rely on. Their study’s starting point was previous research in a related foxglove species, which suggested that biosynthesis occurred through pregnenolone—a molecule often dubbed the “mother of all steroid hormones” because it serves as the precursor for major human steroid hormones like testosterone, progesterone, and estrogen.

Through comparative analysis of the two plant species, the research team identified candidate genes involved in cardenolide biosynthesis. Cardenolide structures in these plants exhibited both overlapping and divergent profiles. Consequently, cross-referencing genetic information from these plants, particularly regarding gene expression related to metabolite formation, played a pivotal role in pinpointing the enzymes responsible for pregnenolone production, as explained by study leader Prashant Sonawane, who oversees the project group specializing in “Steroidal Specialized Metabolism in Plants” within the Department of Natural Product Biosynthesis.

Furthermore, the scientists grappled with the challenge of pinpointing where these desired metabolites accumulated within different plant parts. “Tissue-specific localization of cardenolides was pivotal in utilizing genetic datasets to select 13 candidate genes. Comparing these datasets across various plants helped us narrow down the candidate genes for further characterization,” elucidates Prashant Sonawane.

Ultimately, the team identified two enzymes from the cytochrome P450 family 87A that catalyze the conversion of cholesterol and phytosterols into pregnenolone in both foxglove and Calotropis procera. This represented the initial step in the cardenolide biosynthetic pathway in these distantly related plants and marked the first reported enzymatic function for this subfamily of cytochrome P450.

To validate their findings, the researchers genetically modified Arabidopsis thaliana plants to produce more CYP87A enzymes. These modified plants accumulated unusually high levels of pregnenolone. Additional confirmation of CYP87A enzymes’ involvement in pregnenolone and cardenolide formation came from genetically modified foxglove plants lacking CYP87A enzymes in their leaves, resulting in significantly reduced pregnenolone and cardenolide production. This initiative also marked the establishment of the first stable transformation system for modifying foxglove plants to study specialized metabolites.

The research team’s journey is far from complete as they continue to unravel the subsequent steps in cardenolide formation in various plant species. The biosynthetic pathway remains intricate and lengthy, but with the application of state-of-the-art sequencing, bioinformatics, and metabolomics methods across diverse plant species, they anticipate solving this puzzle in the near future, as stated by Prashant Sonawane.

Plants are valuable sources of numerous pharmaceutical compounds. However, extracting these natural products remains a complex and often unsustainable process. Sarah O’Connor, who leads the Department of Natural Product Biosynthesis at the Max Planck Institute for Chemical Ecology, emphasizes that the discovery of enzymes such as CYP87A could revolutionize the development of sustainable platforms for producing high-value plant compounds by utilizing other plants for their biosynthesis.

Source: Max Planck Society

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