Molecular Determinants of Carbocation Cyclisation in Bacterial Monoterpene Synthases

• Published in: Chembiochem : a European journal of chemical biology 2022-03, Vol.23 (5), p.e202100688-n/a
• Main Authors: Leferink, Nicole G. H., Escorcia, Andrés M., Ouwersloot, Bodi R., Johanissen, Linus O., Hay, Sam, Kamp, Marc W., Scrutton, Nigel S.
• Format: Article
• Language: English
• Subjects: Amino acids; Aromatic compounds; Bacteria; Catalytic Domain; Cineole; Computational chemistry; Computer applications; Cyclization; enzyme catalysis; Eucalyptol; Intermediates; Linalool; molecular dynamics; protein engineering; Reaction mechanisms; Residues; synthetic biology; terpenoids
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.202100688

Monoterpene synthases are often promiscuous enzymes, yielding product mixtures rather than pure compounds due to the nature of the branched reaction mechanism involving reactive carbocations. Two previously identified bacterial monoterpene synthases, a linalool synthase (bLinS) and a cineole synthase (bCinS), produce nearly pure linalool and cineole from geranyl diphosphate, respectively. We used a combined experimental and computational approach to identify critical residues involved in bacterial monoterpenoid synthesis. Phe77 is essential for bCinS activity, guiding the linear carbocation intermediate towards the formation of the cyclic α‐terpinyl intermediate; removal of the aromatic ring results in variants that produce acyclic products only. Computational chemistry confirmed the importance of Phe77 in carbocation stabilisation. Phe74, Phe78 and Phe179 are involved in maintaining the active site shape in bCinS without a specific role for the aromatic ring. Phe295 in bLinS, and the equivalent Ala301 in bCinS, are essential for linalool and cineole formation, respectively. Where Phe295 places steric constraints on the carbocation intermediates, Ala301 is essential for bCinS initial cyclisation and activity. Our multidisciplinary approach gives unique insights into how carefully placed amino acid residues in the active site can direct carbocations down specific paths, by placing steric constraints or offering stabilisation via cation‐π interactions. Active site residues in terpene synthases each have a unique and precise function in guiding the reactive carbocation intermediates to the final product. Here we used a combined experimental and computational approach to unravel the role of several active site residues in bacterial monoterpene synthases. These residues are often not conserved even in closely related enzymes demonstrating the importance of a tailored active site for each terpene product.