Exploring evolutionary use of single residue switches for alternative product outcome in class II diterpene cyclases

Class II diterpene cyclases (DTCs) define the widespread labdane-related diterpenoids. These are particularly prevalent in plants due to the requisite production of gibberellin (GA) phytohormones, specifically from gene duplication and neofunctionalization of the relevant DTC. Alteration of product outcome can be predicted/engineered to some extent by changes in the ancestral histidine-asparagine catalytic base dyad found in the ent-copalyl pyrophosphate (ent-CPP) synthases (CPSs) involved in GA biosynthesis. It has been shown such changes can switch product outcome in CPSs, with substitution of alanine for either leading to incorporation of water - i.e., production of 8α-hydroxy-ent-labda-13-en-15-yl pyrophosphate (ent-LPP), while replacing the histidine with tyrosine leads to production of a rearranged product - i.e., ent-kolavenyl pyrophosphate (ent-KPP). Indeed, native ent-KPP synthases from dicots with such substitution have been found, and restoration of the ancestral residue results in production of ent-CPP. Observation of a similar ent-KPP synthase and, strikingly, an ent-LPP synthase with serine in place of the asparagine, along with another DTC with such substitution but still producing ent-CPP, was recently made in non-seed plants. Here the role of these substitutions was examined by ancestral residue restoration. Notably, while this led to the production of ent-CPP in the first two concordant cases, in the latter incongruent DTC this had little effect. This presumably reflects extended adaptation, consistent with its more distant phylogenetic relationship to those from GA biosynthesis. This demonstrates both the utility but also limitations of the ability of changes to the ancestral catalytic base dyad to affect product outcome.