Protonation-Triggered Carbon-Chain Elongation in Geranyl Pyrophosphate Synthase (GPPS)

Geranyl pyrophosphate synthase (GPPS) is responsible for the formation of geranyl pyrophosphate (GPP), a key intermediate which has the potential to derive numerous functionally and structurally diverse groups of terpenoid natural products via the head-to-tail assembly of two isoprenoid building blocks (dimethylallyl diphosphate, DMAPP; isopentenyl diphosphate, IPP) in the initial step of carbon-chain elongation during isoprenoid biosynthesis. Elucidating the detailed catalytic mechanism in GPPS is of significant interests as it will stimulate the development of new technology in generating novel natural productlike scaffolds. It has been known that the catalytic reaction involves three sequential steps, namely “ionization–condensation–elimination”, but the exact catalytic mechanism has remained controversial since the 1970s. By employing Born–Oppenheimer density functional quantum mechanics (B3LYP/6-31­(+)­G*)/molecular mechanics dynamics simulations, here we suggest that GPPS adopts a protonation-induced catalytic mechanism, in which there are two key points different from previously hypothesized mechanisms. The first point is the protonation of DMAPP which is essential in the initial “ionization” process but was not considered in previous mechanisms. The second point is the stereoselectivity of proton transfer (H_S) from IPP to H76 residue in the final “elimination” step as identified in our simulations, in contrast to the proton transfer from IPP (H_R) to DMAPP in previous hypotheses. Moreover, the free energy barrier of the whole assembly reaction is predicted to be 18.8 ± 0.6 kcal/mol, in agreement with the experimental value of 18.0 kcal/mol. Furthermore, the catalytic roles of the two Mg²⁺ ions at the bottom of the active site are also discussed, and key residues (K44, R47, R94, R95, K180, K235, and E73 around DMAPP and IPP) responsible for the stabilization of transition states, intermediates, and/or product are clarified. These findings can assist site-directed mutagenesis experiments in protein engineering as well as inhibitor designs.