Probing of the plasticity of the active site in pinene synthase elucidates its potential evolutionary mechanism.

Terpenes form a class of highly diverse natural products. The diversity of terpenes is created by terpene synthases. During the reaction, carbocation intermediates form and their rearrangement could lead to the formation of various products. Terpene synthases determine the final product profile by controlling the conformation of the intermediate or stabilizing the carbocation. Pinene synthase is a monoterpene synthase catalyzing the cyclization of geranyl pyrophosphate (GPP) to form pinene. Our study suggests that by mutating the aromatic residue F482 to Ala, Val, Ile and Thr, the enzyme can be converted to sabinene synthase, with more than 90% of its total terpene products becoming sabinene, which indicates the aromaticity of this residue is essential for stabilizing the pinyl carbocation. We also identified a mutation S491A that could cause an about 29% increase in the overall activity of the enzyme without altering its produce selectivity. Molecular dynamic simulation indicates this mutation could decrease the flexibility of the enzyme when it forms a complex with the pinyl carbocation. Our study suggests the active pocket of pinene synthase has a certain level of plasticity, making it relatively easy to change the product selectivity or overall activity. This property could have an important implication in the evolution of terpene synthases and thereby terpene diversity, as by changing a few residues an enzyme could synthesize a completely different terpene product in a significant amount, which allows selection to take place.