Fraser, Nicholas
Description
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...[Show more] 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.
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