Ipsdienol dehydrogenase (IDOLDH): A novel oxidoreductase important for Ips pini pheromone production

High substrate specificity of ipsdienol dehydrogenase (IDOLDH), a short-chain dehydrogenase fromIps pinibark beetles

“…This led to the suggestion that other enzymes 'tune' the enantiomeric ratios of ipsdienol produced by myrcene hydroxylases. One candidate enzyme, ipsdienol dehydrogenase (IDOLDH, GenBank JN653323.1), is produced in a pattern consistent with other pheromone-biosynthetic enzymes and stereospecifically oxidizes (À)-ipsdienol to ipsdienone (2-methyl-6-methylene-2,7-octadien-4-one) (8). The expression pattern and activity of IDOLDH strongly suggested that it serves to produce pheromonal ipsdienol.…”

“…IDOLDH is a member of the short-chain family of alcohol dehydrogenases (SDRs) with activity that places it in the E.C.1.1.1 group (8). These enzymes have very low sequence conservation, but retain the common Rossman fold tertiary structure (rev.…”

“…Recombinant IDOLDH was produced from a baculoviral vector in Sf9 (insect) cell culture, which has proven highly reliable for studies of pheromone biosynthetic enzymes (58, 17), essentially as described previously (8). Assays were done using recombinant IDOLDH recovered in three different ways from infected Sf9 cells: from crude culture media (8), cell homogenates (crude, infected cell lysate), or cell homogenates partially purified with (NH 4 ) 2 SO 4 (see below).…”

“…Assays were done using recombinant IDOLDH recovered in three different ways from infected Sf9 cells: from crude culture media (8), cell homogenates (crude, infected cell lysate), or cell homogenates partially purified with (NH 4 ) 2 SO 4 (see below). Enzyme preparations were stored at 4 C no longer than a month until enzyme assays were done.…”

“…The samples were mixed by vortexing for 30 s and further mixed on a rotator for 16 h or overnight at 4 C. The samples were then centrifuged at 10,000 Â g for 20 min at 4 C, and solid (NH 4 ) 2 SO 4 was added to the supernatant to a final concentration of 40% (40PPIDOLDH) and 60% as described above. The distribution of IDOLDH in pellets and supernatants at different (NH 4 ) 2 SO 4 concentrations was determined by sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDSPAGE) and western blotting using an IDOLDH-specific polyclonal antibody as described previously (8). To test for storage temperature stability, all PPIDOLDH was flash frozen in liquid nitrogen and stored at 4, À20 or À80 C for a week, then thawed at room temperature and assayed using the standard assay as described below.…”