The evolutionary history and modern diversity of triterpenoid cyclases

Cyclic terpenoids are a class of lipid compounds containing immense structural and functional diversity, with many cyclic triterpenoids acting as membrane components, and, for a few well-studied molecules, regulators of membrane physical properties and spatial organization. Cyclic terpenoids are also readily preserved as terpane fossils, such as steranes and hopanes, which form a rich record of the evolution of life on Earth. Formation of the multiple ring structure of all cyclic terpenoids is catalyzed by terpenoid cyclase enzymes, among which are whole clades of proteins – many from environmental metagenomes and uncultured organisms – whose substrates and products are completely unknown. We investigate the function of these divergent cyclases through biochemical assays, and the evolutionary processes that produced them by testing and applying a variety of evolutionary models. We find a deep divergence between the diterpenoid cyclases and all other cyclases. This roots the triterpenoid cyclase tree, demonstrating reciprocal monophyly between squalene-hopene cyclases and sterol cyclases, which is consistent with their simultaneous first occurrence in the geological record. Through a simple test of evolutionary rate shifts, we find positive selection on the enzyme active site on the squalene-hopene cyclase stem. Finally, by testing the activity of divergent cyclases for a variety of substrates, we find a group of stem sterol cyclases from bacteria that synthesize arborinols, two of which produce the molecular precursor to a Permian “orphan biomarker.” Together, our data present an evolutionary framework for triterpenoid cyclases that can inform both the biochemical potential of these proteins and their products’ occurrence in the geological record.