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Monoterpenes (C10 isoprenoids) are a structurally diverse group of natural compounds that are attractive to industry as flavours and fragrances. Monoterpenes are produced from a single linear substrate, geranyl diphosphate, by a group of enzymes called the monoterpene cyclases/synthases (mTC/Ss) that catalyse high-energy cyclisation reactions involving unstable carbocation intermediates. Efforts towards producing monoterpenes via biocatalysis or metabolic engineering often result in the formation of multiple products due to the nature of the highly branched reaction mechanism of mTC/Ss. Rational engineering of mTC/Ss is hampered by the lack of correlation between the active site sequence and cyclisation type. We used available mutagenesis data to show that amino acids involved in product outcome are clustered and spatially conserved within the mTC/S family. Consensus sequences for three such plasticity regions were introduced in different mTC/S with increasingly complex cyclisation cascades, including the model enzyme limonene synthase (LimS). In all three mTC/S studied, mutations in the first two regions mostly give rise to products that result from premature quenching of the linalyl or α-terpinyl cations, suggesting that both plasticity regions are involved in the formation and stabilisation of cations early in the reaction cascade. A LimS variant with mutations in the second region (S454G, C457V, M458I), produced mainly more complex bicyclic products. QM/MM MD simulations reveal that the second cyclisation is not due to compression of the C2-C7 distance in the α-terpinyl cation, but is the result of an increased distance between C8 of the α-terpinyl cation and two putative bases (W324, H579) located on the other side of the active site, preventing early termination by deprotonation. Such insights into the impact of mutations can only be obtained using integrated experimental and computational approaches, and will aid the design of altered mTC/S activities towards clean monoterpenoid products.

Experiment and Simulation Reveal How Mutations in Functional Plasticity Regions Guide Plant Monoterpene Synthase Product Outcome