Electrostatic Control of Chemistry in Terpene Cyclases

Electrostatic interactions play a major role in stabilizing transition states in enzymes. A crucial question is how general this electrostatic stabilization principle is. To address this point, we study a key common C-C bond formation step in a family of enzymes that is responsible for the biosynthesis of 60% of all natural products. In these terpene cyclases, we have previously shown that the enzymes gain chemical control by raising the energy of initial carbocation intermediates along the reaction coordinate to bypass the formation of unwanted side products. Here we employ hybrid quantum mechanics molecular mechanics free energy simulations to show that this energy tuning is achieved by modulation of electrostatic interactions. The tempering of electrostatic interactions allows enzymatically directed chemical control that slows down the reaction temporarily by introducing thermodynamic and activation barriers. We show that this electrostatic control in terpene cyclases is achieved by a unique binary active site architecture with a highly charged region flanked by a hydrophobic region. In the charged region, negatively and positively charged moieties are arranged in approximately a layered manner relative to the carbocation binding pocket, with alternating negative and positive layers. We suggest that this active site architecture can be utilized for rational design.