Determinants of Selectivity for the Formation of Monocyclic and Bicyclic Products in Monoterpene Synthases

Abstract

Monoterpene synthases (MTSs) catalyze the initial committed step in the biosynthesis of monoterpenes. MTSs control challenging reactions that involve highly reactive carbocations through exquisite steric and electrostatic confinement, in some cases with remarkable product specificity and enantioselectivity. Using two well-characterized MTSs as models, (4S)-(−)-limonene synthase (LMNS) and (+)-bornyl diphosphate synthase (BPPS), we implemented an iterative approach that involves comparative atomistic simulations and experimental testing of wild-type enzymes and thirty-six variants to identify the mechanistic underpinnings of selectivity. Free energy simulations indicate that a common reaction intermediate, the α-terpinyl cation (ATC), preferentially adopts one of two different conformations in LMNS and BPPS, thus leading to the formation of monocyclic monoterpenes in the former and bicyclic products in the latter. An assessment of the ATC binding free energy in LMNS, BPPS, and variants revealed that nonbonded interactions with active site residues explain the propensity of the ATC to assume a favored conformation that is consistent with the experimentally determined reaction outcome. The free energy of the ATC in different environments (active sites of LMNS, BPPS, and variants) correlates strongly with the ratio of monocyclic to bicyclic products in model MTSs.

Decoding Catalysis by Terpene Synthases

“…This is particularly important for the longer substrates of C 15 and above, but even GPP (C 10 ) possesses sufficient functionality for two cyclizations. The mechanistic basis for mono- or bicyclic selectivity was explored in detail for the model mTSs Ms(−)-LimS and (+)-bornyl diphosphate synthase (BPPS) . Limonene is monocyclic, and bornyl diphosphate is bicyclic.…”

Bioinspired Catalyst Design Principles: Progress in Emulating Properties of Enzymes in Synthetic Catalysts

“…The scaffold also may induce specific ligand geometries and configurations of the reactants not otherwise observed. , In essence, it creates an environment around the active site that can control the atomic distribution of charge and functionality. Variances in redox potentials, the orientation and delivery of reactants, and the energetics of the transition state are all influenced by the local environment surrounding the active site and are typical examples of outer-sphere impacts. ,,− …”

“…In short, the scaffold creates a confined space (steric and electrostatic) in which reactions occur, a space that can lead to variations in chemical reactivity (the excess chemical potential as defined for porous compounds) compared to unconstrained systems. The scaffold also may induce specific ligand geometries and configurations of the reactants not otherwise observed. , In essence, it creates an environment around the active site that can control the atomic distribution of charge and functionality. Variances in redox potentials, the orientation and delivery of reactants, and the energetics of the transition state are all influenced by the local environment surrounding the active site and are typical examples of outer-sphere impacts. ,,− …”

Biochemical basis for the formation of organ-specific volatile blends in mint

“…Despite the large body of published work on the biosynthesis of functionalized monoterpenoids in mint (Lange and Srividya, 2019), surprisingly, only two MTSs have been functionally characterized: (-)-limonene synthase from spearmint (Colby et al, 1993) and (-)-b-pinene synthase from M. longifolia CMEN 585 (Kim et al, 2022). These MTSs are involved in the biosynthesis of p-menthane monoterpenoids, such as (-)-menthol of peppermint, (-)-carvone of spearmint, and (+)-pulegone of M. longifolia CMEN 585 (all derived from (-)-limonene) and (+)-pinocarvone (derived from (-)-b-pinene) (Figure 1).…”

“…We recently reported on the characterization of an MTS from M. longifolia CMEN 585 that generates (-)-b-pinene as its main product (Kim et al, 2022), which is an intermediate in the biosynthesis of (+)-pinocarvone (Figure 1). Both volatiles were emitted by stems but not from roots, which correlated well with gene expression patterns (the transcript for (-)-b-pinene synthase (Ml_39080) was expressed at relatively high levels in stems but barely detectable in roots of M. longifolia CMEN 585 and CMEN 584; Vining and Pandelova, 2022) (Supplemental Tables S4, S5).…”