EPINZINGIBERENE SYNTHASE: STRUCTURE, MECHANISM AND A TEMPLATE FOR DESIGN OF BIOACTIVE CHEMICAL SPACE UNDERPINNING INSECT OLFACTION
Investigating a new class of sesquiterpene synthases that use (Z,Z)-farnesyl diphosphate as a substrate and can generate novel whitefly semiochemicals
Monday, February 1, 2016 - 16:45
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 as substrates, site directed mutagenesis of the EZS 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, because the products of EZS, 7-epizingiberene and (R)-curcumene, are repellents for whiteflies, major global agricultural and horticultural crop pests, our novel synthetic biological products will have superior properties to the natural products.
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.
Specific objectives include:
- Production of the purified enzymes using an established laboratory bacterial system;
- Develop novel chemistry to produce synthetic ZZ-FDP analogues that can be added to enzyme preparations;
- Convert synthetic substrate analogues to 7-epizingiberene and (R)-curcumene analogues using unmodified EZS and elucidate the catalytic mechanism;
- 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;
- Use electrical recordings of the antennae of insects (electrophysiology), and laboratory behavioural assays, to measure the activity of the generated analogues with whitefly species.
A. (2E,6E)-FDP 1 and (2Z,6Z)-FDP2.
B. Homology model of EZS based on taxadiene synthase.
C. A close-up of the active site region of EZS showing residues targeted for mutagenesis.
D. 7-epizingiberene (3), and (R)-curcumene (4).
Cardiff University - Professor Ruedi Allemann (Principal Investigator); Dr David Miller