Two pockets in the active site of maize sesquiterpene synthase TPS4 carry out sequential parts of the reaction scheme resulting in multiple products

One of the most interesting features of terpene synthases is their ability to form multiple products with different carbon skeletons from a single prenyl diphosphate substrate. The maize sesquiterpene synthase TPS4, for example, produces a mixture of 14 different olefinic sesquiterpenes. To understand the complex TPS4 reaction mechanism, we modeled the active site cavity and conducted docking simulations with the substrate farnesyl diphosphate, several predicted carbocation intermediates, and the final reaction products. The model suggests that discrete steps of the reaction sequence are controlled by two different active site pockets, with the conformational change of the bisabolyl cation intermediate causing a shift from one pocket to the other. Site-directed mutagenesis and measurements of mutant activity in the presence of (E,E)- and (Z,E)-farnesyl diphosphate as substrates were employed to test this model. Amino acid alterations in pocket I indicated that early steps of the catalytic process up to the formation of the monocyclic bisabolyl cation are probably localized in this compartment. Mutations in pocket II primarily inhibited the formation of bicylic compounds, suggesting that secondary cyclizations of the bisabolyl cation are catalyzed in pocket II.