Structure and Function Studies of Insect Carboxylesterases

Abstract

Insect carboxylesterases (CBEs) have great importance in insect biology and play a crucial role in the ever-increasing occurrence of insecticide resistance. This resistance to insecticides (e.g. organophosphates) leads to downstream effects on agriculture and to human health (through reduced crop production and the spread of insect-borne diseases). Structural and functional studies of insect CBEs are vital for combating insecticide resistance, finding new potential targets and in providing insight into the evolution of new protein function. The work described in this thesis contributes to the overall understanding of the structure, function and evolution of insect CBEs.

Chapter 1 is the introduction into the structure, function and evolution of insect CBEs. In particular, the classification of CBEs is discussed, as well, the current understanding of the role CBEs play in insect species. Chapter 2 describes the evolution of new oligomeric structure in the CBE αE7 from Lucilia cuprina and shows that higher order thermostable oligomers can be selected for during evolution. This work affirms the importance of oligomers in evolution to maintain or increase protein stability and demonstrates structure/activity trade-offs that are observed throughout enzyme evolution.

Chapter 3 describes structural studies into the CBE esterase-6 (EST6) from Drosophila melanogaster - a CBE that has shown to be important for the reproductive success of Drosophila species. The enzyme has a unique active site entry for insect CBEs, which results in a narrow and shortened active site. Docking simulations in combination with kinetic analyses show that the enzyme is a probable odorant-degrading enzyme. It also indicates that EST6 does not directly interact with the sex pheromone 11-cis vaccenyl acetate (cVA), which contradicts the previously held belief that it is the main substrate of the enzyme.

The evolution of CBEs involved in insecticide resistance in several species is discussed in Chapter 4. α-esterase orthologs from different dipteran species were analysed to discern important factors for the evolution of qualitative insecticide resistance and the signifiance of evolutionary contingency. A large variation in binding and turnover for the organophosphate compound was observed with the introduction of the Gly137Asp mutation into the orthologs. Given the similarity in the predicted structures of the orthologs, it suggests that second and third shell mutations are important in mediating the catalytic effects conferred by the Gly137Asp mutation in the orthologs.

Chapter 5 discusses the development and testing of new inhibitors to treat the symptoms of Alzheimer’s disease, through molecular docking simulations. Galanthamine derivatives showed no significant binding to AChE, however, the tested marinoquinoline derivatives displayed a large variation in affinity for AChE. Chapter 6 summarises the conclusions that these studies have allowed us to make regarding the structures, functions and evolution of insect CBEs. The chapter finishes with a discussion of future work that could be undertaken to extend these findings and further develop our understanding of this important enzyme family.