Taming the Reactivity of Monoterpene Synthases To Guide Regioselective Product Hydroxylation

Abstract

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.

Redesigning the Molecular Choreography to Prevent Hydroxylation in Germacradien-11-ol Synthase Catalysis

“… 31 , 43 To avoid any possible water-activating role of the residue at 313, Q313 was also changed to the nonpolar A; previously, a water-activating role for an active-site Asn residue was indicated (N305 in the hydroxylating bacterial 1,8-cineole synthase, bCinS). 36 …”

The Impression of a Nonexisting Catalytic Effect: The Role of CotB2 in Guiding the Complex Biosynthesis of Cyclooctat-9-en-7-ol

“…18,22−31 (5) Specific positioning of an active site water molecule or acids/ bases that allow regioselective substrate (de)protonation, hydroxylation, or epoxidation. 12,31,32 One of the challenges in studying the detailed chemistry occurring in TPS is the lack of crystal structures with bound ligands in a catalytically competent, fully closed active site state. 12,33 In the case of the monoterpene TPS bornyl diphosphate synthase, several feasible crystal structures exist, 34 which facilitated extensive in silico studies.…”

Insights into the enzymatic catalytic mechanism of bCinS: the importance of protein conformational change

“…2 The plausible enzyme catalysis mechanism of bCinS from substrates (NPP/GPP) to the product 1,8-cineole. The pathways with red and blue arrows are proposed based on the previous crystal structures 19 and the isotope 27 as well as chiral GC 28 experiments, respectively.…”

“…This indicated the feasibility of the reaction proceeding though the 'S' configuration as demonstrated by experiments. 27,28 Then, the active water added to the C7 carbocation. Instead of forming the (S)-terpinyl hydronium ion, the stable neutral intermediate (S)-terpineol was spontaneously yielded by proton transfer (H1) of the water molecule to the proton acceptor N305 (Fig.…”

“…27 Recently, Leferink and coworkers have also verified the (S)-isomer preference of bCinS by chiral GC analysis, which is inconsistent with the previous verdict from crystals. 28 Thus, this has evoked our interest in probing the detailed reaction mechanism catalyzed by bCinS.…”