Investigating conserved aromatic residues in ent-copalyl pyrophosphate synthases required for gibberellin phytohormone biosynthesis

Terpene synthases and cyclases catalyze carbocation cascade reactions, which have been hypothesized to be directed, in part, by aromatic residues via stabilization of specific intermediates through cation-π interactions towards specific product outcomes. Included in this are class II diterpene cyclases (DTCs), which are particularly widespread due to their role in initiating biosynthesis of gibberellin (GA) phytohormones but also function in production of a vast range of more specialized (labdane-related) diterpenoids. Indeed, the ent-copalyl pyrophosphate synthases (CPSs) required for GA biosynthesis are then conserved in all plants, with certain plant-associated bacteria that also produce GA containing potentially distantly related such CPSs as well. Building on the structure determined for the CPS from Arabidopsis thaliana (AtCPS), sequence comparison reveals that all seven aromatic residues in the active sites are conserved, suggesting these may play important roles in the catalyzed reaction. The role of these aromatic residues in directing product outcome was then examined via a series of substitutions for each in two representative examples, one from plants (AtCPS) and the other bacteria (EtCPS from Erwinia tracheiphila). Strikingly, substitution with even aliphatic residues had relatively little effect on product outcome, indicating more general structural roles for these aromatic groups. Accordingly, the role of aromatic residues in directing the carbocation cascade reactions catalyzed by at least such CPSs, if not also terpene cyclases and perhaps even synthases, requires additional evidence beyond simple presence in the active site, even when conserved.