Epizingiberene synthase: structure, mechanism and a template for design of bioactive chemical space underpinning insect olfaction

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Terpene synthases together generate the largest and most diverse family of natural products from a small pool of achiral substrates. They are hence a paradigm in the study of enzymatic catalysis performing arguably the most complex single-step reactions known in nature. They catalyse a highly intricate carbocationic reaction cascade involving changes in connectivity and hybridisation of up to half the carbon atoms of the substrate, often distinguishing between intermediate species of very similar energy with phenomenal selectivity. Until recently the sesquiterpenes were all thought to originate from just one substrate (E,E-farnesyl diphosphate, EE-FDP) but recently a new class of sesquiterpene synthases that employ ZZ-FDP exclusively as substrate has been discovered. We will perform the first mechanistic investigation of this exciting new class of enzyme using 7-epizingiberene synthase (EZS). Use of modified ZZ-FDPs, site directed mutagenesis and structural studies will reveal more completely the complex interplay between substrate and catalyst for this important family of enzymes, closing a clear gap in our knowledge of terpene biosynthesis.Moreover the products of EZS, 7-epizingiberene and (R)-curcumene, are repellents for whiteflies, major global agricultural and horticultural crop pests. External chemical signals (semiochemicals) mediate many interactions between organisms. These are typically low molecular weight lipophilic compounds such as sesquiterpenes. Upon release, such signals can act by modifying either the behaviour or the development of recipient organisms. Structural analogues would solve many of the problems associated with their use since they may have enhanced efficacy and stability. However, until recently no rational approach has emerged by which to conduct SAR studies since receptor systems for semiochemicals are extremely selective, since they must select from an enormous diversity and concentration range of external chemicals, (cf. receptor systems within organisms for which analogues can be designed). In a previous study, we demonstrated that the bioactive space of analogues of germacrene D, a sesquiterpene that is released by crop plants under stress and which repels aphid populations, could be dictated by use of germacrene D synthase (GDS). This supported our hypothesis that FDP analogues accepted by GDS would lead to products retaining the required structural features for activity. The generality of this approach developed by us has yet to be fully tested however and so as important added value to this project we will use EZS to generate a further generation of bioactive whitefly repellents.Success in this work will ensure the continued leading international presence of UK synthetic biology and lead to environmentally benign approaches to crop protection and food security.Thus, the overall aims of the project are to fully characterise a member of a new class of sesquiterpene synthases with the added benefit of producing biologically active analogues of 7-epizingiberene and (R)-curcumene, representing a second generation of synthetic semiochemicals whose chemical space is dictated by the constraints of biosynthesis. The specific objectives include: 1) production of the purified enzymes using an established laboratory bacterial system; 2) develop novel chemistry to produce synthetic ZZ-FDP analogues that can be added to enzyme preparations; 3) convert synthetic substrate analogues to 7-epizingiberene and (R)-curcumene analogues using unmodified EZS and elucidate the catalytic mechanism; 4) Perform structural studies and site-directed mutagenesis for elucidation of the mode of action of EZS plus use modified EZSs to convert synthetic an extended range of ZZ-FDP substrates to analogues; 5) use electrical recordings of the antennae of insects (electrophysiology), and laboratory behavioural assays, to measure the activity of the generated analogues with whitefly species.