Taming the Reactivity of Monoterpene Synthases To Guide Regioselective Product Hydroxylation

• Published in: Chembiochem : a European journal of chemical biology 2020-04, Vol.21 (7), p.985-990
• Main Authors: Leferink, Nicole G. H., Ranaghan, Kara E., Battye, Jaime, Johannissen, Linus O., Hay, Sam, Kamp, Marc W., Mulholland, Adrian J., Scrutton, Nigel S.
• Format: Article
• Language: English
• Subjects: Binding Sites; Catalytic Domain; Cineole; Cyclization; Cyclohexane Monoterpenes - chemistry; Cyclohexane Monoterpenes - metabolism; enzyme catalysis; Eucalyptol - chemistry; Eucalyptol - metabolism; Flavors; Fragrances; Hydroxylation; Industrial plants; Intramolecular Lyases - genetics; Intramolecular Lyases - metabolism; Molecular dynamics; Molecular Dynamics Simulation; Monoterpenes - chemistry; Monoterpenes - metabolism; Monoterpenoids; Mutagenesis, Site-Directed; Mutation; Natural products; Pharmaceutical industry; protein engineering; Quantum mechanics; Stereoisomerism; Streptomyces - enzymology; synthetic biology; terpenoids; Terpineol; Water - chemistry; Water - metabolism
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.201900672

Monoterpenoids are industrially important natural products with applications in the flavours, fragrances, fuels and pharmaceutical industries. Most monoterpenoids are produced by plants, but recently two bacterial monoterpene synthases have been identified, including a cineole synthase (bCinS). Unlike plant cineole synthases, bCinS is capable of producing nearly pure cineole from geranyl diphosphate in a complex cyclisation cascade that is tightly controlled. Here we have used a multidisciplinary approach to show that Asn305 controls water attack on the α‐terpinyl cation and subsequent cyclisation and deprotonation of the α‐terpineol intermediate, key steps in the cyclisation cascade which direct product formation towards cineole. Mutation of Asn305 results in variants that no longer produce α‐terpineol or cineole. Molecular dynamics simulations revealed that water coordination is disrupted in all variants tested. Quantum mechanics calculations indicate that Asn305 is most likely a (transient) proton acceptor for the final deprotonation step. Our synergistic approach gives unique insight into how a single residue, Asn305, tames the promiscuous chemistry of monoterpene synthase cyclisation cascades. It does this by tightly controlling the final steps in cineole formation catalysed by bCinS to form a single hydroxylated monoterpene product. Promiscuous chemistry of cyclisation cascades: A synergistic experimental and computational approach revealed that Asn305 tightly controls the water attack and subsequent cyclisation step in the reaction cascade of bacterial cineole synthase, resulting in 95 % pure cineole via the (S)‐(−)‐α‐terpineol intermediate.