Proteomic studies of the pheromone biosynthetic pathway in the bark beetle, Ips pini, and molecular modeling of an insect-specific aldehyde oxidative decarbonylase, CYP4G2
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Authors
Abbott, Nicole L.
Issue Date
2013
Type
Dissertation
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Abstract
Bark beetles have had devastating economic and environmental impacts in both
the United States and Canada in the last decade. Male pioneer pine engraver beetles, Ips
pini, find a host tree and release an aggregation pheromone, ipdienol. Ipsdienol is
produced de novo in midgut tissue and is released through the frass to signal other beetles
to initiate a mass attack and colonize a tree. Both feeding and juvenile hormone III
(JHIII) treatment stimulate pheromone production. Studies of the genes involved in the
pheromone biosynthetic pathway have showed that mevalonate pathway and downstream
genes are upregulated. However, there is not necessarily a direct relationship between
transcript and protein levels, and this work used proteomic studies to examine protein
levels in midgut tissue. 2D-DIGE and ProQ Diamond phosphoprotein staining were used
to examine the expression of proteins and phosphorylated proteins involved in the
pheromone producing midgut tissue. The results show a lack of a linear relationship
between the high transcript levels previously reported and an increase in protein
expression. In addition, several key proteins involved in pheromone production in
midgut tissue appear to be phosphorylated when male beetles were treated with juvenile
hormone III to induce pheromone production.
Cytochromes P450 are a diverse superfamily of enzymes that are found in
organisms ranging from bacteria to animalia. The P450 family has a broad substrate
range and catalyzes reactions involving both xenobiotics and endogenous substrates.
Musca domestica and Drosophila melanogaster each contain a closely related P450,
CYP4G2 and CYP4G1, respectively. Their known function is to oxidatively
decarbonylate long-chain aldehydes to produce hydrocarbons. Because of the difficulties in expressing and assaying CYP4G2, molecular modeling was used to gain a better
understanding of this enzyme. The molecular model of CYP4G2 was constructed using
homology modeling and molecular mechanics with Sybyl8.0, AMBER 99_7 force field,
using PDB1DT6 (CYP2C5) as a template.
Methyl branches are inserted early in fatty acid synthesis during methyl-branched
hydrocarbon production. They are located toward the methyl end of the fatty acid and
resulting aldehyde. It is hypothesized that methyl branches closer to the carbonyl group
of the aldehyde could hinder substrate binding and/or could create an energy barrier when
the substrate or product travel through the substrate channel. Thus, it is hypothesized that
insects evolved enzyme systems to put the methyl branch on the methyl end of the fatty
acid and aldehyde. The model was used with Sybyl8.0, AMBER99_7 force field to
calculate the individual energies of the substrates and products (octadecanal, nheptadecane, 4-methyloctadecanal, 4-methylheptadecane, and 3-methylheptadecane) as
they traveled through the solvent accessible channel. Although experimental assays
need to be done to confirm this model, the analysis of the difference of van der Waals
energies between methyl-branched and straight chain substrates suggest a possible energy
barrier that methyl branched substrates and products must overcome in traveling through
the substrate channel.
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