Structural diversity of isoprene synthases in mosses from multiple terpenoid synthase lineages.

Isoprene, a C5 hydrocarbon emitted by various land plants, serves as a protective molecule under heat stress. The moss Calohypnum plumiforme isoprene synthase (ISPS) catalyzes isoprene formation from dimethylallyl diphosphate and contains two aromatic residues (Y393 and F615) at the bottom of the active site that restrict the cavity size to accommodate the smaller C5 substrate. Here, we show that these residues are critical determinants of substrate size selectivity. Substitution of the two residues with alanine (Y393A and F615A) expanded the active site and exhibited diterpene synthase activity, generating ent-pimaradienes from ent-copalyl diphosphate. Further mutagenesis of the catalytic motif yielded ent-kaurene, demonstrating stepwise functional conversion from diterpene synthase to ISPS activity. Screening of ISPS genes across mosses identified two structurally unrelated enzyme classes: typical diterpene synthase-type ISPSs (C. plumiforme, Pohlia nutans) and microbial-type terpene synthase (TPS)-like ISPSs (Polytrichum commune, Leucobryum juniperoideum), the latter representing a previously unrecognized ISPS scaffold. Microbial-type TPS-like ISPSs of P. communehave two aromatic residues (W151 and F288), as in the case of C.plumiformeISPS, to restrict the cavity size. Substitution experiments expanding the active site in ISPS of P. commune (W151A and F288A) determined the production of ocimenes as monoterpene synthase activity. These results define the molecular basis of substrate specificity in TPSs and demonstrate that structurally divergent enzyme families can independently acquire ISPS activity.