Monoterpenoid metabolism by bark beetle cytochromes P450

Insect cytochromes P450 are physiologically important in the synthesis and degradation of endogenous compounds and the detoxification of exogenous substances including host plant chemicals and insecticides. Thus, cytochromes P450 present a unique target for controlling insects, including bark beetles (Coleoptera: Scolytidae).;Here I report the cDNA isolation, characterization, and the first functional expression of three P450s, Ips pini CYP9T2, I. confusus CYP9T1, and Dendroctonus ponderosae CYP6BW1, from the Coleoptera. Quantitative real-time PCR experiments showed that feeding on host phloem induced CYP9T2 expression in males, but not females, and that basal expression levels are highest in male midguts, similar to other I. pini pheromone-biosynthetic genes. Microsomes from recombinant Sf9 cells producing CYP9T2 and house fly (Musca domestica ) NADPH-cytochrome P450 reductase converted myrcene to mostly ( 4R)-(-)-ipsdienol, an important aggregation pheromone component for western North American I. pini. These results are consistent with CYP9T2 encoding a myrcene hydroxylase that functions near the end of the pheromone-biosynthetic pathway.;I. confusus CYP9T1, an ortholog to CYP9T2, converts myrcene to a similar enantiomeric ratio of ipsdienol as CYP9T2. Since I. pini and I. confusus use contrasting enantiomeric versions of the aggregation pheromone component ipsdienol, these data support the idea that additional biosynthetic enzymes are required following myrcene hydroxylation to achieve the critical quantity and chirality of pheromonal ipsenol and ipsdienol used by different Ips species.;Ipsdienol production by CYP9T2 and IcCYP9T1 increased with incubation time and increasing myrcene concentration. Both enzymes also catalyzed the conversion of alpha-pinene to trans-verbenol and cis-verbenol. The molecular structure of CYP9T2 was investigated with a homology model constructed using the crystal structure CYP102 (P450 BM-3) as the template. The three-dimensional model shows that the CYP9 protein shares a highly conserved structural core and similarly positioned sequence motifs with other cytochromes P450. Substrate docking indicated that myrcene and alpha-pinene fit well in the rather small, hydrophobic active sites of both CYP9T2 and IcCYP9T1.;Nine unique P450s from D. ponderosae midguts and fat bodies were isolated and the expression patterns in response to feeding for four of them were compared to 3-hydroxy-3-methylglutarylcoenzyme A reductase (HMG-R), which is involved in frontalin production. The most abundant mRNA, representing CYP6BW1, was localized predominantly in the midgut, and feeding on host phloem modestly induced CYP6BW1 expression in males and females. Preliminary functional assays using microsomes of Sf9 cells co-expressing baculoviral-mediated recombinant CYP6BW1 and house fly reductase showed that CYP6BW1 converted alpha-pinene to the female aggregation pheromone component, trans-verbenol.;The data in this Dissertation provide a solid foundation for further exploration of P450 substrate and product profiles, and their biological roles. They also provide significant insight into the evolution of pheromone biosynthesis from resin detoxification, thus improving our understanding of bark beetle-host tree interactions. These enzymes have potential application in the development of new modes of controlling bark beetles and may also be useful for biotechnological production of monoterpenoids.