Simulation-guided redesign of terpene synthase product outcome Marc Van der Kamp University of Bristol, UK Terpenoids are the largest and most diverse class of natural products with a wide array of possible uses, including as flavour compounds, insect repellents and medicine (e.g. anti-cancer or anti-malarial activity). Terpene synthases are responsible for producing the many highly complex and structurally diverse terpenes from acyclic isoprenyl diphosphate substrates. Most are Class I terpene synthases with the same overall structure and several conserved features 1 . Mechanistically, these terpene synthases first capture the diphosphate substrate with its prenyl chain in a largely hydrophobic active site cavity and then split the substrate into diphosphate and a highly reactive carbocation. This carbocation can then undergo a series of cyclisations, hydride- and methyl shifts, and/ or proton transfers, prior to being quenched through proton abstraction or hydroxylation. Here, we demonstrate how subtle differences in the active site template and interactions can steer the carbocation cascade towards a specific final terpene product 2,3 , with a focus on the nature of carbocation quenching. These insights are obtained through detailed atomistic simulations, and we provide examples of how simulation-guided mutagenesis allows targeted production of complex non-hydroxylated terpenoids in hydroxylating terpene synthases 4 . We further discuss our ongoing work of introducing controlled hydroxylation in non-hydroxylating terpene synthases – a particular challenge. Our work demonstrates how combining simulation and experiment can provide routes to the biosynthesis of specific complex terpenoid compounds. References 1. D. W. Christianson.Structural and Chemical Biology of Terpenoid Cyclases. Chem. Rev. 2017 , 117 ,11570–11648,DOI: 10.1021/acs.chemrev.7b00287 2. N. G. H. Leferink, K. E. Ranaghan, V. Karuppiah, A. Currin,M. W. van der Kamp, A. J. Mulholland and N. S. Scrutton. Experiment and Simulation Reveal How Mutations in Functional Plasticity Regions Guide Plant Monoterpene Synthase Product Outcome, ACS Catalysis 2018 , 8 , 3780-3791. DOI: 10.1021/acscatal.8b00692 3. N. G. H. Leferink, K. E. Ranaghan, J. Battye, L. O. Johannissen, S. Hay,M. W. van der Kamp, A. J. Mulholland and N. S. Scrutton.Taming the Reactivity of Monoterpene Synthases To Guide Regioselective Product Hydroxylation, ChemBioChem 2020 , 21 , 985-990. DOI: 10.1002/cbic.201900672 4. P. L. Srivastava, A. M. Escorcia, F. Huynh, D. J. Miller, R. K. Allemann and M. W. van der Kamp. Redesigning the Molecular Choreography to Prevent Hydroxylation in Germacradien-11-ol Synthase Catalysis, ACS Catalysis 2021 , 11 , 1033-1041. DOI: 10.1021/acscatal.0c04647.
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