Insecticide resistance is a global concern that threatens human health and agricultural productivity. Understanding the molecular basis of resistance will help to manage future insecticide use to ensure that effective, safe and inexpensive pest control is available. In the Australian sheep blowfly Lucilia cuprina, a single mutation (Gly137Asp) in the αE7 carboxylesterase gives rise to resistance by converting the enzyme into an organophosphate (OP) hydrolase. This emergence of new activity provides a unique opportunity to investigate the molecular basis for enzyme evolution. In this thesis, I investigated the structure, function, evolution and inhibition of αE7. Chapter two describes the role of structural diversity in the function of wild type αE7. I applied new methods for extracting information about structural diversity from X-ray diffraction data to explore the changes in structure that accompany high affinity OP binding in αE7. In chapter three, I investigated the molecular basis for the evolution of catalytic OP detoxification in the blowfly. I determined the structure of the Gly137Asp variant by X-ray crystallography, which, along with molecular dynamics simulations and enzyme activity assays, revealed the role of Asp137 in the new catalytic mechanism. The new sidechain is disordered, and potentially only displays a fraction of its catalytic potential. Chapter four explores this catalytic potential through the laboratory-directed evolution of αE7 for increased OP hydrolase activity. I performed detailed kinetic and structural analysis of the evolutionary trajectory and characterized the structural changes responsible for the 8000-fold increase in OP hydrolase activity. The analysis unmasked a hidden, catalytically relevant, conformation of the active site. Furthermore, the results revealed the role of conformational diversity in the evolutionary optimization of αE7 and highlight the challenges to satisfying the competing demands of substrate binding and catalysis in
the tightly packed environment of an enzyme’s active site. This work establishes that only a fraction of the evolutionary potential of αE7 has been explored in nature. In chapter five, I combined structural knowledge of αE7 with a computational screen to discover new potent and selective inhibitors of αE7. These compounds, based on a boronic acid scaffold, act as synergists to reduce the amount of OP required to kill L. cuprina by up to 16-fold, and abolish resistance. The broad-spectrum potential for
the compounds as a new class of synergist was demonstrated by their low toxicity to animals and their ability to potentiate OP insecticides against another common insect pest, the peach-potato aphid Myzus persicae. These compounds represent a solution to OP resistance as well as to environmental concerns regarding overuse of OPs, allowing significant reduction of use without compromising efficacy. More broadly, this thesis makes contributions to characterizing structural protein heterogeneity using X-ray diffraction, to understanding the molecular basis of enzyme evolution and to the use of in silico screens for the discovery of enzyme
inhibitors. The results from this thesis will assist the of control insect pests and the management of insecticide resistance
