Engineering of plant isoprenyl diphosphate synthases for development of irregular coupling activities

Irregular monoterpenes perform specialized functions in different groups of organisms. Among insect pheromones, various irregular monoterpene structures are found displaying branched, cyclopropane and cyclobutane skeletons. These compounds can be applied to control pest populations in a strictly targeted and therefore environmentally friendly way. Introduction of the enzymes catalysing formation of irregular terpenes into plant metabolic networks can ensure precise and efficient synthesis of insect pheromones.
The broad distribution of irregular monoterpenes within the higher taxonomic ranks is in the same time restricted to a limited number of genera and species. It leads to the assumption that the enzymes producing irregular monoterpenes have evolved repeatedly from their regular counterparts that must possess considerable structural plasticity.
To gain insight into the process of development of irregular activities, we performed mutagenesis on two regular isoprenyl diphosphate synthases (IDS) of plant origin. Both of them have closely related analogues performing non-head-to-tail coupling of two dimethylallyl diphosphate (DMAPP) units. Farnesyl diphosphate synthase from Tanacetum cinerariifolium (TcFPPS) shares 70.6 % identity with chrysanthemyl diphosphate synthase from the same plant species (TcCPPS). Neryl diphosphate synthase from Solanum lycopersicum (SlNPPS) is to 40.6 % identical to lavandulyl diphosphate synthase from Lavandula x intermedia (LiLPPS). TcFPPS and SlNPPS represent two different classes of regular IDS, trans- and cis-IDS, respectively.
Wild type TcFPPS shows only regular IDS activity. Exchange of phenylalanine 89 of TcFPPS for isoleucine conferred an irregular activity on this enzyme. The mutant supplied with DMAPP as the only substrate produces two products, although at a low level. The second analysed enzyme, wild type SlNPPS, was able to catalyse the coupling of two DMAPP units and formed two irregular monoterpene diphosphates; their structures were elucidated by the NMR analysis of their dephosphorylation products. As expected based on homology with LiLPPS, one of the alcohols is lavandulol. The second compound represents the first example of an irregular cyclobutane monoterpene produced by a plant enzyme, the trans-isomer of planococcol. The irregular activity of SlNPPS constitutes 8% of its regular activity and is revealed only if the enzyme is supplied with DMAPP in absence of isopentenyl diphosphate (IPP). The exchange of asparagine 88 for histidine increased the irregular and decreased the regular activity of SlNPPS substantially changing the ratio between them to 37%.
The obtained results prove that the regular IDS of trans- and cis-classes are promising starting points for protein engineering aiming the development of irregular activities and leading to novel monoterpene structures.

Recommended Articles