Myrcene Hydroxylases do not Determine Enantiomeric Composition of Pheromonal Ipsdienol in Ips spp.

Abstract

Myrcene (7-methyl-3-methylene-1,6-octadiene) hydroxylation is likely one of the final reactions involved in the production of the Ips spp. (Coleoptera: Scolytidae) aggregation pheromone components, ipsdienol (2-methyl-6-methylene-2,7-octadien-4-ol) and ipsenol (2-methyl-6-methylene-7-octen-4-ol). To gain insight into the evolution of pheromone production, we isolated a full-length cDNA from the pinyon ips, Ips confusus (LeConte), that encodes a pheromone-biosynthetic cytochrome P450, I. confusus CYP9T1 (IcCYP9T1). The recovered cDNA is 1.70 kb, and the open reading frame encodes a 532 amino acid protein. IcCYP9T1 is 94% identical to the pine engraver, Ips pini (Say), CYP9T2 ortholog that hydroxylates myrcene. Quantitative real-time PCR experiments showed that IcCYP9T1, as does CYP9T2, has an expression pattern similar to other pheromone-biosynthetic genes in I. pini. Basal expression levels were higher in males than females, and expression was significantly induced in male, but not in female, anterior midguts by feeding on host phloem. Microsomes, prepared from Sf9 cells co-expressing baculoviral-mediated recombinant IcCYP9T1 and house fly (Musca domestica) NADPH-cytochrome P450 reductase, converted myrcene to ~85%-(R)-(-)-ipsdienol. These results are consistent with IcCYP9T1 encoding a myrcene hydroxylase that functions near the end of the pheromone-biosynthetic pathway. Since the I. confusus pheromone blend contains >90%-(S)-(+)-ipsdienol, these results confirm further that Ips spp. myrcene hydroxylases do not control the final ipsdienol enantiomeric blend. Other enzymes are required following myrcene hydroxylation to achieve the critical quantity and enantiomeric composition of pheromonal ipsenol and ipsdienol used by different Ips spp.

Functional expression of Ips paraconfusus cytochromes P450 exhibiting conspicuous up-regulation with feeding on host phloem.

“…More recently, an analogue of this gene in Ips confusus LeConte, IcCYP9Tl, was expressed in SF9 cells. The resulting enzyme performed the same function (Sandstrom et al, 2008). Considering how many bark beetles not only utilize identical or similar aggregation pheromones, but also colonize hosts with similar monoterpene complements, it stands to reason that similar genes will be found in other species.…”

“…Generally, N-termini will encode a signal for one location or the other; microsomal P450s generally have hydrophobic N-terminal residues followed by a basic region and several proline residues, whereas mitochondrial P450s have N-termini with interspersed positive residues and a cleavable site (reviewed by Omura, 1999;Neve & IngelmanSundberg, 2008). The above two peptide sequence patterns can be sufficiently recognizable to predict localization of some P450s (Sandstrom et al, 2006(Sandstrom et al, , 2008, but motif-recognizing software can also be employed (e.g. Huber et al, 2007).…”

“…The following are examples of functionally characterized P450 enzymes, but there are several cases where bark beetle representatives have yet to be elucidated beyond conspicuous expression levels with varying treatments. However, a few bark beetle P450s have been either functionally characterized or otherwise implicated in pathways related to colonization success (Sandstrom et al, 2006(Sandstrom et al, , 2008Huber et al, 2007).…”

“…Thirty minutes is the generally accepted incubation time for such assays (Wen et ah, 2003;Sandstrom et al 2006Sandstrom et al , 2008, but one hour was also tested in case of inefficient electron transfer or a slow substrate turnover rate. Results did not differ with increasing incubation time.…”

“…Table 3.1 summarizes these findings. At this point, only one pheromone-producing P450 has been confirmed (CYP9T2 and its ortholog IcCYP9Tl) (Sandstrom et al, 2006(Sandstrom et al, , 2008. Given that host-colonizing bark beetles are exposed to many xenobiotics, that they produce different enantiomeric ratios of pheromones, and the diversity of the pheromone components themselves, there are many detoxifying and pheromone-producing P450s that have yet to be elucidated.…”