PNNL

The Science 

Biological molecules can exhibit handedness, or chirality, which can strongly influence their properties and functions. But precisely and consistently converting achiral molecules into chiral molecules is a challenge. A combined experimental and computational approach revealed that monoterpene synthases establish product handedness before the first stereocenter forms. By selectively stabilizing either a right- or left-handed helical pose of the achiral substrate geranyl diphosphate (GPP), the enzyme biases the reaction toward a particular stereochemical outcome. In contrast, the handedness of the downstream intermediate linalyl diphosphate (LPP) is not a reliable predictor of the final products. Across enzymes and variants, the preferred GPP pose is the stronger predictor of the product handedness.

The Impact 

Valuable molecules in biochemistry, including pharmaceuticals, are often chiral, with different forms often exhibiting markedly different properties. Developing synthetic routes to convert achiral starting materials into molecules with a specific stereochemistry is essential for discovering and manufacturing useful chemicals. This work reveals an upstream “preorganization” principle for controlling enzyme stereochemistry, providing design rules for engineering catalysts that make desired chiral chemicals from simple, accessible precursors.

Summary 

Many biological molecules occur in left- and right-handed forms, which can have very different properties. To create chiral terpenes, monoterpene synthases must solve a fundamental stereochemical problem. They begin with GPP, an achiral molecule, yet the terpenes they produce have a defined handedness. This study shows that the enzyme biases the product handedness before the first stereocenter is formed. The active site molds GPP into one of two helical poses—right- or left-handed. The pose that is preferentially stabilized strongly predicts whether the enzyme produces predominantly left- or right-handed products. In contrast, the configuration of the later intermediate LPP does not consistently predict the final stereochemical outcome.

The researchers established this relationship by combining stereochemical product measurements for six enzymes and selected variants with molecular dynamics simulations, binding free-energy calculations, and a reduced thermodynamic-kinetic model. Across this enzyme set, the calculated preference for a GPP pose tracked the experimentally observed product distribution. The finding identifies substrate preorganization as an upstream control element for enzyme stereoselectivity and suggests a practical engineering strategy: redesign active-site interactions to favor one GPP pose over the other rather than trying to redirect the reactive carbocation cascade after ionization has occurred. This principle could enable more predictive design of biocatalysts for producing selected chiral terpenes from accessible precursors.

Contact 

Simone Raugei, Pacific Northwest National Laboratory, simone.raugei@pnnl.gov 

Funding 

Support for N.S. and B.M.L. was provided by the U.S. Department of Energy (DOE), Office of Science, Basic Energy Sciences program, Division of Chemical Sciences, Geosciences, and Biosciences (grant DE-SC0001553). Support for H.K. and S.R. was provided by the DOE, Office of Science, Basic Energy Sciences program, Division of Chemical Sciences, Geosciences, and Biosciences. Computational work was performed in part using the Molecular Sciences Computing Facility in the Environmental Molecular Sciences Laboratory, a DOE user facility located at Pacific Northwest National Laboratory (PNNL). Battelle operates PNNL for the DOE under contract DE-AC05-75RL01830.