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Proteomic studies of the pheromone biosynthetic pathway in the bark beetle, Ips pini, and molecular modeling of an insect-specific aldehyde oxidative decarbonylase, CYP4G2
AuthorAbbott, Nicole L.
AdvisorBlomquist, Gary J.
Biochemistry and Molecular Biology
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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.