PNNL

Abstract

Monoterpene synthases (MTSs) catalyze the first committed step in the biosynthesis of monoterpenes, which play critical roles in the interactions of plants with their environment and are also widely used as ingredients for the flavor and fragrance industry. Monoterpene synthases control challenging reactions in a selective and stereospecific manner without the intervention of cofactors. Using two well-characterized MTSs, (4S)-(-)-limonene synthase (LMNS) and (+)-bornyl diphosphate synthase (BPPS), as models, we implemented an iterative approach that involved comparative atomistic simulations and experimental testing of wild-type enzymes and selected mutants to identify the mechanistic underpinnings of their specificity. Free energy calculations using force-field- and hybrid QM/MM-based molecular dynamics simulations indicate that a common reaction intermediate, the a-terpinyl cation (ATC), preferentially adopts two different conformations in LMNS and BPPS, which lead to the formation of monocyclic monoterpenes in the former and bicyclic products in the latter. An assessment of the ATC absolute binding free energy in LMNS, BPPS, and selected variants revealed that non-bonded interactions with active site residues explain the propensity of the ATC to assume a favored conformation. The integration of atomistic simulations with experimental testing of wild-type enzymes and multiple variants allowed us to assign specific functions to active site residues (N345 and M458 in LMNS, I344 and I457 in BPPS) for controlling the ratio of monocyclic to bicyclic products in model MTSs, explaining the experimentally determined reaction outcome. This study is laying the foundation for predicting the product specificity of plant MTSs.