Mountain pine beetle (MPB; Dendroctonus ponderosae) pheromone component biosynthesis, cytochromes P450 and monoterpene metabolism in bark beetles
Biochemistry and Molecular Biology
AltmetricsView Usage Statistics
Bark beetles successfully attack host trees by metabolizing host monterpenes efficiently and producing aggregation pheromones. Investigating enzymes involved in pheromone biosynthesis and monoterpene metabolism may identify unique molecular targets and provide information for developing new means to deal with bark beetle infestations. The three major mountain pine beetle (MPB, Dendroctonus ponderosae) pheromone components are frontalin, exo-brevicomin and trans-verbenol. Although these components were identified several decades ago, none of their biosynthetic pathways are known in detail and none of the involved enzymes are identified or characterized. This dissertation explores biochemical details of two important MPB pheromone components: exo-brevicomin and trans-verbenol, and bark beetle monoterpene metabolism in the context of specific enzymes. In vivo assays revealed that exo-brevicomin was predominantly produced in male fat bodies, and not in other tissues or by females, and that fat bodies catalyzed the conversion of decanoic acid to nonen-2-one, confirming that (6Z)-non-6-en-2-one is likely derived from fatty acid. Fat bodies converted (6Z)-non-6-en-2-one to 6,7-epoxynonan-2-one, the direct precursor of exo-brevicomin, and (6Z)-non-6-en-2-ol. The epoxide was stable under physiological conditions. These results implicate a cytochrome P450 and cyclase in the terminal steps of exo-brevicomin biosynthesis and (6Z)-non-6-en-2-ol may be the precursor of (6Z)-non-6-en-2-one.Two novel enzymes are implicated in exo-brevicomin production: a cytochrome P450, CYP6CR1, and a novel short chain dehydrogenase, ZnoDH. Their mRNA profiles were consistent with exo-brevicomin production suggests their coordinate regulation. Both enzymes were expressed in Sf9 cells for enzyme assays. Recombinant ZnoDH oxidized (6Z)-non-6-en-2-ol to the corresponding methyl-ketone, (6Z)-non-6-en-2-one, which serves as the substrate for CYP6CR1. CYP6CR1 converted (6Z)-non-6-en-2-one to 6,7-epoxynonan-2-one. These results suggest both CYP6CR1 and ZnoDH are in the exo-brevicomin biosynthetic pathway. Furthermore, two alternative pathways yielding production of the C9 precursor from a 10:1-fatty acid are discussed in light of these new data. While a direct decarboxylation of a â-ketoacyl-CoA intermediate has been suggested, evidence presented here supports oxidative decarbonylation of an unsaturated C10 aldehyle to produce 3-nonene, followed by hydroxylation to (6Z)-non-6-en-2-ol and oxidation by ZnoDH to (6Z)-non-6-en-2-one. Other cytochromes P450 were investigated to explore the evolutionary link between pheromone production and resin detoxification. Enzyme assays showed CYP6DH1 and its paralog, CYP6DH2, likely have complementary roles. CYP6DH1 produced verbenol from á-pinene, but did not hydroxylate other monoterpenes and was not induced by its substrates. Therefore, it likely functions as a pheromone (trans-verbenol) biosynthetic enzyme. In contrast, CYP6DH2 had a broad substrate range, suggesting a role in resin detoxification. CYP6DH1 most likely evolved from the detoxification enzyme, CYP6DH2, by duplication followed by genetic drift. Similarly, I. pini CYP9T2 had essentially the same substrate profile as CYP6DH2 even though CYP9T2 has been confirmed as a pheromone biosynthetic enzyme. Both enzymes converted the same substrates into different products, suggesting that different substrate binding regions may be involved in orienting the substrates. These results support the evolutionary mechanism that CYP9T2 or its ancestor originally worked as a detoxification enzyme, but it is now dedicated to pheromone production.