Origin and functional differentiation of (E)-β-ocimene synthases reflect the expansion of monoterpenes in angiosperms

The acquisition of new metabolic activities is major force driving evolution. We explored how new functions have arisen diverging terpene synthases (TPSs) from the perspectives of gene family expansion and the evolutionary adaptability of proteins. Monoterpenoids are diverse natural compounds that can be divided into cyclic or acyclic skeleton forms according to their chemical structure. We demonstrate, through phylogenetic reconstructions and genome synteny analyses, that the acyclic monoterpene synthases (mTPSs) (E)-β-ocimene synthases appear to have arisen several times in independent lineages during plant evolution. Bioinformatics analyses and classical mutation experiments identified the four sites (I388-F420-S446-F485) playing important roles in the neofunctionalisation of mTPSs. Incubation of neryl diphosphate (NPP) with Salvia officinalis 1, 8-cineole synthase (SCS) and mutated proteins show that these four sites obstruct the isomerisation of GPP. QM/MM MD simulations of models of SCS, SCSY420F/I446S, and SCSN338I/Y420F/I446S/L485F with (3R)-linalyl diphosphate ((3R)-LPP) suggest that mutations change the configuration of the intermediate to obtain new activities. These results provide new perspectives on the evolution of mTPSs, explain the convergent evolution of (E)-β-ocimene synthases at the molecular level, and identify key residues to control the specificity of engineered mTPSs.