A conserved mechanism affecting hydride shifting and deprotonation in the synthesis of hopane triterpenes as compositions of wax in oat

Abstract

Triterpenoids are biologically active metabolites synthesized from a common linear precursor catalyzed by 2,3-oxidosqualene cyclases (OSCs) to form diverse triterpenoid skeletons. OSCs corresponding to many discovered triterpene alcohols in nature have not been functionally and mechanistically characterized due to the diversity of chemical structures and complexity of the cyclization mechanism. We carried out a genome-wide investigation of OSCs from Avena strigosa and discovered two triterpene synthases, namely, AsHS1 and AsHS2, using a Nicotiana benthamiana expression system. These synthases produce hopenol B and hop-17(21)-en-3β-ol, which are components of surface wax in oat panicles and sheathes, respectively. We demonstrated that substitutions of two to three amino acid residues in AsHS1 with corresponding residues from AsHS2 allowed it to be completely converted into a hop-17(21)-en-3β-ol synthase. AsHS2 mutants with a substitution at site 410 could synthesize hopenol B alone or mixed with a side product isomotiol. The combined quantum mechanics and molecular mechanics calculation demonstrated that the side chain size of the residue at site 410 regulated the relative orientations between the hopyl C22 cation and Phe257, leading to a difference in deprotonation positions through providing or not providing cation–π interaction between the aromatic ring of F257 and the carbocation intermediate. A similar mechanism could be applied to a hopenol B synthase from a dicotyledonous plant Aquilegia. This study provided mechanistic insight into triterpenoid synthesis and discovered key amino acid residues acting on hydride transfer and a deprotonation site to differentiate between hopane-type scaffolds in diverse plant species.