Peeking behind the carbocation: identification of (alternative) catalytic bases in the class II active site of conifer resin acid diterpene synthases.

Abstract

Class II diterpene cyclases (DTCs) initiate biosynthesis of the labdane-related diterpenoids (LRDs), utilizing an acid-base mechanism to catalyze bicyclization of the general diterpenoid precursor (E,E,E)-geranylgeranyl pyrophosphate (1), most often producing the eponymous labdadienyl/copalyl pyrophosphate (CPP, 2). Prominent among the LRDs and terpenoids more generally are the conifer resin acids. The abietaenol synthase from Abies grandis (AgAS), due in part to crystallographic structural analysis, serves as a model for the DTCs initiating resin acid biosynthesis, with such activity having been conserved for over 300 million years. Previous work suggests that a hydrogen-bonded tyrosine-histidine pair in its DTC active site serves as the catalytic base, in part because the substitution of aspartate for the histidine or phenylalanine for the tyrosine leads to the incorporation/addition of water and the production of labda-13-en-8α-ol-15-yl pyrophosphate (LPP, 3). However, the exact identity of the catalytic base in the native reaction, as well as any alternative base(s) enabling the production of 3 and 7-endo-CPP (4) in the histidine to aspartate mutant, remains unknown. Here, the TerDockin computational approach, combining quantum chemical modeling with computational docking, was applied to the AgAS DTC active site. This not only indicated the Tyr hydroxyl group serves as the native catalytic base but also surprisingly found a serine capable of serving as an alternative base for the production of 3 and a tyrosine serving as the alternative base for the production of 4, as supported by mutational analysis in AgAS. This provides mechanistic insight and further validates the TerDockin approach to investigation of these important enzymes.