Isoprenoid Binding and Substrate Channeling in Drimenol Synthase, a Bifunctional Class II Terpene Cyclase-Phosphatase

More than one thousand bifunctional terpene synthases combining prenyltransferase and terpene cyclase activities have been identified in bacteria and fungi, but only a handful of enzymes have been identified that combine terpene cyclase activity with a downstream processing activity. Drimenol synthase from the marine bacterium Aquimarina spongiae (AsDMS) consists of a class II terpene cyclase that converts farnesyl diphosphate into drimenyl diphosphate, and a haloacid dehalogenase-like phosphatase that hydrolyzes drimenyl diphosphate to generate the sesquiterpene alcohol drimenol. The first crystal structure of AsDMS to be reported revealed the architecture of domain assembly as well as dimeric quaternary structure, establishing a structural chemical foundation for cyclization and hydrolysis mechanisms [K. R. Osika, M. N. Gaynes, D. W. Christianson (2025) Proc. Natl. Acad. Sci. U.S.A. 122, e2506584122]. Here, we report crystal structures of the catalytically-inactive double mutant, D33A-D323A AsDMS, complexed with farnesyl diphosphate, geranyl diphosphate, and dimethylallyl diphosphate, which bind in the active sites of both the cyclase and phosphatase domains. Molecular recognition of the diphosphate group dominates binding interactions in both active sites. In the cyclase active site, only farnesyl diphosphate is sufficiently long for its terminal isoprenoid C=C bond to bind adjacent to the catalytic general acid that would initiate the cyclization cascade in the wild-type enzyme. In the phosphatase active site, all isoprenoid diphosphate groups bind similarly, but isoprenoid chain conformations vary. These structures provide a foundation for understanding substrate recognition and catalysis in both active sites. Finally, we present kinetic evidence suggesting that substrate channeling is operative in wild-type AsDMS.