Ipsdienol Dehydrogenase (IDOLDH), a Novel Oxidoreductase Important in the Last Steps of Pheromone Biosynthesis in Ips Spp. (Coleoptera: Scolytinae: Curculionidae)
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Authors
Figueroa-Teran, Rubi D.
Issue Date
2011
Type
Dissertation
Language
Keywords
Bark Beetles , Ipsdienol dehydrogenase , Ips pini , monoterpene dehydrogenase , Pheromone , Short chain dehydrogenase/reductase (SDR)
Alternative Title
Abstract
Ips spp. beetles biosynthesize ipsdienol and ipsenol in different enantiomeric
blends and ratios as pheromones. In order to understand how these beetles evolved the
ability to use the same components to synthesize different pheromone blends, the last
steps of ipsdienol and ipsenol biosynthesis, which are probably catalyzed by
oxidoreductases, must be characterized.
Here I report the isolation of ipsdienol dehydrogenase (IDOLDH) from the three
bark beetles (Coleoptera): western Ips pini (wIDOLDH), I. confusus (IcIDOLDH) and
eastern I. pini (eIpIDOLDH). IDOLDH is the first characterized non-dipteran insect
monoterpene short-chain dehydrogenase/reductase (SDR). Quantitative real-time PCR
experiments showed that wIDOLDH transcript was induced by feeding in male midguts,
the hallmark of pheromone biosynthetic genes. Surprisingly, protein levels were
unaffected by feeding, suggesting other factor(s) control pheromone biosynthesis.
IDOLDH was present only in male midguts, not in females or other tissues. Functional
characterization of wIDOLDH and IcIDOLDH provide the first direct evidence for ipsenol
biosynthesis through ketone intermediates and interconversion of (-)-ipsdienol to
ipsdienone.
WIpIDOLDH oxidized racemic and (-)-ipsdienol to ipsdienone and reduced
ipsdienone to (-)-ipsdienol and ipsenone to (-)-ipsenol, but discriminated against (+)-
ipsdienol as a substrate. IcIDOLDH similarly oxidized (-)-ipsdienol to ipsdienone,
discriminated against (+)-ipsdienol, reduced ipsdienone to ipsdienol (stereochemistry not
determined), and used ipsenone as a substrate. Ongoing studies showed eIpIDOLDH
had similar activities. Kinetic analysis of IDOLDH oxidation of (-)-ipsdienol with NADP⁺
followed the Michaelis-Menton model, indicating this type of analysis is adequate to
characterize IDOLDH catalyzed reactions. The expression profiles and conservation of
activities across three species strongly supports that IDOLDH has evolved specifically
for pheromone biosynthesis in Ips beetles. The functional data indicates that IDOLDH
contributes to, but does not solely control the enantiomeric blend of ipsdienol in Ips spp.
Additionally, ipsenone was not a reduction product of ipsdienone, suggesting an
ipsenone reductase (IDONER) is required for ipsenone biosynthesis.
IDOLDH’s primary structure contains all the critical motifs of an alcohol
dehydrogenase in the SDR superfamily. Primary sequence identity of IDOLDH
isozymes was not as high as expected for sibling species (82%), however the substrate
binding loop was highly identical (99%) and the fact that they retain similar substrate
profiles suggest that the differences are not important in determining function. Primary
sequence comparisons with the human L-3-hydroxyacyl-CoA dehydrogenase type II/
amyloid-β binding alcohol dehydrogenase (hHADH II/ ABAD) showed a much lower
identity (36%) but was still surprisingly high for such diverse organisms with different
substrate preferences. Although the overall architecture of these enzymes is similar the
substrate binding loop regions are completely different, except for the anchor portions,
suggesting that much of the substrate specificity differences are probably found in this
region. This work adds to our overall understanding of monoterpene and insect SDRs.
The data in this dissertation allow the prediction that Ips beetles probably attain
their final pheromone blends by the interplay between pheromone biosynthetic enzymes
including IDOLDH, myrcene hydroxylase, (+)-ipsdienol oxidoreductase (unidentified),
and ipsdienone reductase (unidentified), whose activities are probably controlled by the
redox state of the cell. Additionally, this work provides a solid foundation for future
studies on insect SDRs and their involvement in pheromone biosynthesis. Future work
to identify, model, and kinetically characterize, other pheromone biosynthetic genes will
provide a map for understanding how insects have evolved the ability to tune pheromone
blends, and aid in identification of targets for pest management.
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