Structure, Function and Evolution of Insect Carboxylesterases

Abstract

Insect carboxylesterases (CBEs) have proven to be a highly adaptable family of enzymes that has undergone extensive functional diversification and sequence divergence over a short span of evolutionary time. This makes these enzymes ideal examples to explore the evolutionary processes that lead to the unique functions of enzymes. In this thesis I present two such examples. The first example is addressed in chapters 2 and 3: the evolution of insecticide resistance CBEs. These enzymes are implicated in the most common forms of insecticide resistance, a global issue that threatens both our agricultural productivity and health. While a great deal of work has gone into the identification of insecticide resistance CBEs, there has been little molecular characterization of these enzymes. This is vital to better understand how they function and to allow target-based inhibitor design to combat the resistance they provide. In chapter 2, I describe my attempts to express a large range of insecticide resistance CBEs in Eschericia coli. This is a critical first step in the large-scale expression required for crystallization and full characterization. I identified five insecticide resistance CBEs with sufficient expression for crystallization trials. In chapter 3, I describe the crystallization and characterization of one of these CBEs, CqestB2, which is the most common insecticide resistance CBE in the important disease vector, Culex quinquefasciatus. CqestB2 is the first insecticide sequestration CBE to be structurally characterized. Its structure demonstrates a high similarity to the insecticide target, acetylcholinesterase. Sequence similarity networks of all insect CBEs demonstrated that insecticide resistance CBEs share a level of similarity. This was further emphasized through a structural comparison between CqestB2 and other insect CBEs. Kinetic characterization of CqestB2 supported its role in organophosphate resistance via sequestration. Finally, a comparison between CqestB2 and its naturally occurring isoforms suggests target-based inhibitor design may have broad applicability. The second example is addressed in chapter 4: the evolution of an odorant degrading enzyme (ODE) from a juvenile hormone esterase (JHE) duplication in Drosophila melanogaster. While the evolution of new functions via gene duplication is a widely accepted mechanism, there are relatively few, well characterized examples of this process. The distinct regulation and substrate specificities of these enzymes also provides a unique opportunity to explore the interaction of both structural and regulatory changes in neofunctionalization. A phylogenetic analysis shows that JHEs have been the template to many distinct functional groups of enzymes. Biochemical comparison reveals sufficient promiscuity in the D. melanogaster JHE (DmJHE) to have immediate utility as an ODE. Homology modelling and comparison with known structures of insect JHEs and ODEs revealed similarities and differences that distinguish these groups and suggests key structural changes that explain this example of neofunctionalization. Finally, in chapter 5, I discuss the significance of my research and the insights that these two examples provide to the process of enzyme evolution. The first, the insecticide resistance CBEs provide a critical example of the early stages of enzyme evolution whereby a promiscuous activity results in a novel function. The comparisons drawn between CqestB2 and LcaE7, an insecticide resistance CBE from Lucilia cuprina that utilizes catalytic detoxification, emphasize distinct strategies through which natural evolution selects for novel functions. The second, DmJHE and DmJHE duplication, provides an example of a later stage in the process of neofunctionalization whereby structural and regulatory changes have resulted in two distinct enzymes with unique functions.