Controlling Product Stereochemistry in Class II Diterpene Cyclases

Class II diterpene cyclases (DTCs) catalyze the committed step in production of the large class of labdane‐related diterpenoid natural products, many of which function as important primary and secondary metabolites in plants. Mechanistic studies of DTCs, which bicyclize geranylgeranyl diphosphate (20‐C), have revealed the importance of a variable set of two amino acids comprising a catalytic base dyad. Here, the catalytic base dyads of the closely related DTCs DsCPS (Danshen copalyl diphosphate synthase) and MvPPS ( Marrubium vulgare peregrinol diphosphate synthase) were probed to further elucidate the evolution and mechanisms of these enzymes. Such studies were completed using site‐directed mutagenesis, with the mutated DTCs expressed in an Escherichia coli metabolic engineering system. DsCPS ordinarily produces copalyl diphosphate (CPP), which adopts the chair‐chair conformation. However, upon mutating the catalytic base dyad residues (along with one other neighboring residue) to the amino acids of the catalytic base dyad in MvPPS, the DsCPS mutant produces terpentedienyl diphosphate (TPP). Intriguingly, TPP adopts the chair‐boat conformation, corresponding to the stereochemistry of the MvPPS product. Thus, simply introducing the catalytic base dyad of one DTC into another is sufficient to change product stereochemistry in this instance, illustrating the critical role of these two amino acids. This research provides valuable insight into how specific mutations in closely related DTCs have created the capacity to produce such a wide array of labdane‐related diterpenoids. Support or Funding Information National Institutes of Health