A Hydrophobic Tunnel and a Gatekeeper at the Active Site Entrance in Two Type I Terpene Synthases

The diterpene synthase SxSpS is known to produce spata-13,17-diene from geranylfarnesyl diphosphate (GGPP), but can also convert farnesyl diphosphate (FPP) and GFPP into structurally similar terpenes. Here we demonstrate that SxSpS is even able to produce a triterpene from FFPP. This behavior is explained by a hydrophobic tunnel observed crystallograpghically that is connected to the active site and can accommodate nonreacting spectator isoprenoid units, as demonstrated by extensive docking studies. The tunnel is contoured by three small amino acid residues, and their systematic exchange against larger residues disrupts the acceptance of higher oligoprenyl diphosphates. A similar tunnel in selina-4(15),7(11)-diene synthase (SdS) is blocked by a gatekeeper, but its opening through site-directed mutagenesis leads to formation of a diterpene.