Computational studies of the E3 carboxylesterase from Lucilia cuprina

The Australian sheep blowfly, Lucilia cuprina, has evolved resistance to organophosphate insecticides via a single point mutation in a carboxylesterase (E3). In addition to their use as insecticides, organophosphates have been synthesised as chemical warfare agents, posing a threat to humans. Thus, an efficient organophosphate detoxification agent, such as E3, has potential value as a prophylactic that could break down organophosphates before they cause intoxication. The work in this thesis aims at understanding the mechanism of organophosphate catalysis by E3 to reveal how insecticide resistance evolved and to evaluate whether E3 could be a useful prophylactic to prevent organophosphate poisoning. The mechanism by which the naturally occurring mutant E3-Gly137Asp catalyses the hydrolysis of organophosphates, as predicted through quantum-cluster calculations, is presented here. Whereas the initial phosphorylation of the active site serine (Ser218) occurs in the mutant the same way as in the wild-type enzyme, the results presented here suggest that the enzyme plays two key roles in the second dephosphorylation step. First, the new Asp137 residue in the active site acts as a general base in the initial nucleophilic attack of a water molecule on the phosphorylated serine. Second, the catalytic histidine residue of E3 (His471) acts as a general acid in the dephosphorylation step, donating a proton to the departing phosphodiester. Additionally, the role of the oxyanion in lowering activation energy barriers was identified. The accuracy of computational methods for the prediction of turnover rates of enzymes is assessed. Despite the limitations present in the methods used, practically useful predictions were achieved. vii The in silico binding of a range of different substrates, both carboxylester and organophosphate, to E3 is analysed in the light of experimental data. Predictions are made about possible natural substrates and the potential uses of E3 to degrade organophosphate chemical warfare agents. This is additionally investigated through the use of X-ray crystallographic data. Molecular dynamics simulations have been used to investigate the effects of substrate presence and mutations on the sampling of different rotamers of amino acids within the active site of E3. The implications of these results on enzyme engineering are discussed. It is suggested that the E3- Gly137Asp mutation might be detrimental to the catalytic activity of E3, owing to its sampling of non-productive conformations. The additional mutations that have accumulated in a laboratory-evolved mutant of E3 (Gly137Asp/Lys306Met/Met308Val/Ser470Gly), which has further enhanced catalytic activity, have apparently reduced the sampling of non- productive states. Altogether, this work has utilized a range of computational techniques, from ligand docking to molecular dynamics simulations and quantum chemical simulations, to generate several novel insights into the catalytic mechanism of the recently evolved organophosphate-degrading E3- Gly137Asp mutant from L. cuprina. As part of a larger research program, many of these hypotheses have been tested, and supported, through enzyme kinetics and protein X-ray crystallography. The new knowledge that has been gained from this work will hopefully aid in the further improvement of this enzyme as an organophosphate detoxification agent and in better understanding how insecticide resistance can evolve

Structure and function of an insect α-carboxylesterase (α Esterase 7) associated with insecticide resistance

Insect carboxylesterases from the αEsterase gene cluster, such as αE7 (also known as E3) from the Australian sheep blowfly Lucilia cuprina (LcαE7), play an important physiological role in lipid metabolism and are implicated in the detoxification of or