Scientists Map Hidden Plant Enzymes That Build Nature’s Most Complex Molecules

A large-scale genome analysis has uncovered thousands of hidden enzyme sequences in plants responsible for constructing triterpenes, a class of structurally diverse and biologically active molecules. By mining 599 plant genomes, researchers identified 1,405 oxidosqualene cyclases (OSCs) and experimentally characterized 20 previously unknown enzymes. Their work revealed new triterpene scaffolds, remarkable enzymatic flexibility, and specific amino acid switches that determine product outcomes. This study provides a roadmap for discovering bioactive natural products directly from sequence data.

Unlocking the Chemical Library Hidden in Plant Genomes

Plants produce an immense variety of chemical compounds, many of which serve as pharmaceuticals, fragrances, and defensive metabolites. Among these, triterpenes stand out because of their intricate ring structures and wide range of biological activities. However, despite decades of research, most enzymes that synthesize triterpenes remain unidentified.

The key enzymes in triterpene biosynthesis are oxidosqualene cyclases, which catalyze the complex transformation of a single linear precursor molecule, 2,3-oxidosqualene, into hundreds of cyclic frameworks. These chemical reactions involve intricate carbocation rearrangements, making them among the most elaborate in nature. While chemists have cataloged thousands of triterpene structures, only a few hundred corresponding OSC genes had been experimentally characterized. This gap limited both biochemical understanding and biotechnological application.

The new study addresses this long-standing limitation by systematically mining plant genomes to identify OSC sequences and functionally testing representative enzymes to reveal their chemical capabilities.

Bridging the Knowledge Gap in Natural Product Biosynthesis

The research team focused on solving a fundamental problem in natural product chemistry: connecting molecular structures to their genetic origins. While modern analytical chemistry can detect and identify thousands of plant metabolites, linking these molecules back to their biosynthetic genes has been challenging. OSCs are particularly problematic because small changes in their amino acid sequences can completely alter the folding of the carbocation intermediate, resulting in distinct triterpene skeletons.

To overcome this challenge, the researchers developed a robust computational pipeline capable of identifying OSC-like sequences even from unannotated genomes. They analyzed 599 plant genome assemblies, spanning 387 species, and extracted a high-confidence set of 1,405 OSC proteins. This large dataset represents the most comprehensive map of plant triterpene synthases to date.

Building a High-Throughput Discovery Pipeline

To manage the large-scale sequence data, the researchers used profile hidden Markov models (pHMMs), which recognize conserved sequence motifs characteristic of OSC enzymes. This allowed them to categorize the OSCs into distinct clades, detect lineage-specific expansions, and prioritize divergent sequences that might encode new catalytic activities.

For functional characterization, 20 uncharacterized OSC genes were selected across different plant lineages. These were transiently expressed in Nicotiana benthamiana, a model plant system commonly used for metabolic assays. To ensure sufficient substrate availability, the researchers coexpressed genes that boost the supply of 2,3-oxidosqualene. The resulting triterpenes were extracted, analyzed by gas chromatography–mass spectrometry (GC–MS), and structurally confirmed by nuclear magnetic resonance (NMR) spectroscopy where needed.

This combination of genome mining, profile-based classification, and functional validation created a powerful workflow capable of revealing new enzyme functions at scale.

Discovery of New Chemical Scaffolds and Hidden Enzyme Flexibility

Previously Unknown Natural Molecules

Of the 20 enzymes tested, 16 produced detectable triterpene products, collectively yielding 41 distinct chemical peaks. Several enzymes were highly specific, producing a single major scaffold, while others were multifunctional, generating multiple related structures. Importantly, several of these scaffolds had never been linked to any known enzyme, and at least two were entirely novel compounds that expand the known diversity of plant triterpenes.

The Kalanchoe Breakthrough

Among the most striking findings was the discovery of two previously unknown pentacyclic triterpenes from Kalanchoe fedtschenkoi. The enzyme responsible, designated OSC16, synthesized molecules the researchers named kalanchoeol and spirokalanchoeol. Spirokalanchoeol displayed an uncommon 6,6,6,6,5 ring structure featuring a spiro-linked junction and a rearranged methyl pattern, suggesting a rare 1,3-alkyl shift during its formation. This mechanism is highly unusual in OSC chemistry and illustrates the remarkable catalytic creativity of plant enzymes.

The structural elucidation of these molecules through NMR not only added new scaffolds to natural chemistry but also provided mechanistic clues about how small structural adjustments in enzymes can enable rare carbocation rearrangements.

Multifunctional Enzymes and Evolutionary Plasticity

The study also showed that several enzymes, previously thought to be functionally narrow, exhibited surprising multifunctionality. Some OSCs produced mixtures of structurally diverse triterpenes, indicating that they may function as evolutionary intermediates exploring chemical space. This plasticity suggests that plants evolve new metabolic functions through gene duplication followed by sequence divergence, allowing them to adapt to ecological pressures by diversifying their chemical defenses.

Pinpointing the Molecular Switches That Control Enzyme Products

One of the most remarkable aspects of the study was the identification of specific amino acid changes that alter product outcomes. In an enzyme from Japanese morning glory (OSC9), researchers discovered that a single substitution, S724V, shifted the product profile from mainly euphane-type triterpenes to predominantly tirucallane-type products.

Further mutagenesis revealed that switching the positions or identities of neighboring residues at positions 548 and 549 (for example, F549L and L548F) caused a similar shift in product ratio. These findings demonstrate that very subtle active-site modifications can redirect the cascade of carbocation rearrangements, determining the final molecular scaffold.

This precise mapping of structure–function relationships provides valuable guidance for enzyme engineers who aim to design OSC variants capable of synthesizing specific triterpenes for pharmaceutical or industrial applications.

Implications for Science and Society

1. Accelerating Natural Product Discovery

By combining computational prediction with experimental validation, the study introduces a scalable approach for discovering new enzymes and metabolites. It effectively bridges the gap between genome sequencing and chemical identification, transforming raw sequence data into chemical knowledge.

2. Opportunities in Synthetic Biology

The identification of key residues that control product specificity opens possibilities for rational enzyme design. Modified OSCs could be used to produce valuable triterpenes in engineered microorganisms or plants, facilitating sustainable production of bioactive compounds, including potential drug precursors and natural sweeteners.

3. Evolutionary Insights

The widespread occurrence of lineage-specific OSC expansions suggests that triterpene diversification has played an important role in plant evolution. The ability of plants to fine-tune OSC functions may have contributed to ecological adaptation, influencing interactions with herbivores, pathogens, and pollinators.

4. Advancing Mechanistic Chemistry

The discovery of spirokalanchoeol challenges conventional mechanistic models of triterpene formation and introduces new rearrangement pathways. These observations could inspire synthetic chemists to develop biomimetic strategies for creating complex ring systems that were previously thought inaccessible.

Limitations and Future Directions

Although the study revealed an impressive range of enzymatic activities, it focused primarily on phylogenetically divergent OSCs. The proportion of multifunctional enzymes might therefore be higher in this sample than in the entire OSC family. Some unproductive or silent enzymes may have failed to express properly or required additional plant-specific cofactors not present in the experimental system.

Future work will involve expanding the dataset to include more plant families, applying structural biology to visualize enzyme–substrate interactions, and exploring the ecological functions of newly discovered triterpenes. Computational modeling combined with experimental validation could also illuminate how carbocation dynamics are guided by active-site geometry.

Toward a Complete Map of Plant Chemical Diversity

The integration of bioinformatics, evolutionary biology, and experimental chemistry has provided a new perspective on plant metabolism. The identification of 1,405 high-confidence OSC sequences across 599 genomes establishes a reference framework for triterpene biosynthesis in the plant kingdom. By making the sequence data and validated enzymes publicly available, the researchers have created a foundation for community-driven exploration of natural product diversity.

This work illustrates how genome data can be transformed into practical biochemical understanding. As genome sequencing becomes routine, such integrated pipelines will make it possible to uncover the enzymatic origins of thousands of unknown plant metabolites, guiding the discovery of new bioactive compounds and advancing synthetic biology.

Conclusion: A Genomic Blueprint for Nature’s Chemical Innovation

The study demonstrates that the complexity of plant chemistry is not beyond reach. By mining hundreds of genomes, systematically classifying OSC enzymes, and functionally characterizing a targeted set, the researchers revealed hidden scaffolds, mechanistic flexibility, and precise amino acid determinants that shape triterpene diversity.

These findings transform our understanding of natural product biosynthesis, linking genomic data directly to molecular architecture. More importantly, they mark a new era where computational exploration and experimental validation converge to decode the chemical potential of life.

The research was published in Nature Chemical Biology on October 6, 2025.