Cyclization mechanism of monoterpenes catalyzed by monoterpene synthases in dipterocarpaceae

Monoterpenoids are typically present in the secretory tissues of higher plants, and their biosynthesis is catalyzed by the action of monoterpene synthases (MTSs). However, the knowledge about these enzymes is restricted in a few plant species. MTSs are responsible for the complex cyclization of monoterpene precursors, resulting in the production of diverse monoterpene products. These enzymatic reactions are considered exceptionally complex in nature. Therefore, it is crucial to understand the catalytic mechanism of MTSs to elucidate their ability to produce diverse or specific monoterpenoid products. In our study, we analyzed thirteen genomes of Dipterocarpaceae and identified 38 MTSs that generate a variety of monoterpene products. By focusing on four MTSs with different product spectra and analyzing the formation mechanism of acyclic, monocyclic and bicyclic products in MTSs, we observed that even a single amino acid mutation can change the specificity and diversity of MTS products, which is due to the synergistic effect between the shape of the active cavity and the stabilization of carbon-positive intermediates that the mutation changing. Notably, residues N340, I448, and phosphoric acid groups were found to be significant contributors to the stabilization of intermediate terpinyl and pinene cations. Alterations in these residues, either directly or indirectly, can impact the synthesis of single monoterpenes or their mixtures. By revealing the role of key residues in the catalytic process and establishing the interaction model between specific residues and complex monoterpenes in MTSs, it will be possible to reasonably design and engineer different catalytic activities into existing MTSs, laying a foundation for the artificial design and industrial application of MTSs.