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

Abstract

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.

An automated pipeline for the screening of diverse monoterpene synthase libraries

“…Here we applied a monoterpenoid screening pipeline which employs a GC-MS based automated pipeline utilising a liquid handling robotic platform for the HTP screening of mTC/S variant libraries in vivo 24 . We targeted 16 amino acid residues that are part of three previously identified conserved plasticity regions 8 in a (-)-α-pinene synthase variant (VAR3-PinS) that produces an equal mixture of linear, monocyclic and bicyclic monoterpenoid products (Fig. 2).…”

“…To address the numbers problem in directed evolution 25 , the NBT degenerate codon (12 codons, 11 amino acid residues) was selected for introduction at the 16 target positions. The mostly polar and hydrophobic residues encoded by NBT correspond to the residues naturally found in the plasticity regions of the plant mTC/S enzyme family 8 , which limits the search to the parts of the sequence space that encode functional variants and avoid areas of non-functional variants. Using the pipeline, approximately 1000 colonies were screened, and over 65 unique sequences with associated product profiles were detected.…”

“…The amino acid residues in the three plasticity regions targeted in this study are indicated. Residues at positions 339 and 558 (in grey) were not targeted as they are highly conserved among plant mTC/S 8 . ( b ) Relative product profiles of native PinS and VAR3-PinS (data obtained from Leferink et al .…”

En route to terpene natural products utilizing supramolecular cyclase mimetics

“…[9][10][11][12][13][14] Such studies have provided insight into how cyclase enzymes exert control over the cyclization outcome, via specic interactions between active site moieties and carbocations. 9,11,14,15 The enzyme, however, not necessarily has to intervene throughout the whole cascade reaction; some steps can be driven by the intrinsic reactivity of the cationic intermediates. 16 Nevertheless, in solution the tail-to-head cyclization mode of class I enzymes proved to be very challenging to reproduce.…”

Taming the Reactivity of Monoterpene Synthases To Guide Regioselective Product Hydroxylation

“…Full product profiles are shown in Figure and Table S3. Interestingly, only the N305A and N305C variants resulted in reasonably active enzymes; the N305D, Q and L variants each produced monoterpenoid titres of <1 mg L org −1 , which suggests that bCinS does not show a high degree of functional plasticity, unlike plant mTC/S, including CinS_Sf, where Asn338 can be replaced by many other residues resulting in active variants with alternative product profiles …”