Metal ions control product specificity of isoprenyl diphosphate synthases in the insect terpenoid pathway

Abstract

Isoprenyl diphosphate synthases (IDSs) produce the ubiquitous branched-chain diphosphates of different lengths that are precursors of all major classes of terpenes. Typically, individual shortchain IDSs (scIDSs) make the C 10 , C 15 , and C 20 isoprenyl diphosphates separately. Here, we report that the product length synthesized by a single scIDS shifts depending on the divalent metal cofactor present. This previously undescribed mechanism of carbon chainlength determination was discovered for a scIDS from juvenile horseradish leaf beetles, Phaedon cochleariae. The recombinant enzyme P. cochleariae isoprenyl diphosphate synthase 1 (PcIDS1) yields 96% C 10 -geranyl diphosphate (GDP) and only 4% C 15 -farnesyl diphosphate (FDP) in the presence of Co 2+ or Mn 2+ as a cofactor, whereas it yields only 18% C 10 GDP but 82% C 15 FDP in the presence of Mg 2+. In reaction with Co 2+ , PcIDS1 has a K m of 11.6 μM for dimethylallyl diphosphate as a cosubstrate and 24.3 μM for GDP. However, with Mg 2+ , PcIDS1 has a K m of 1.18 μM for GDP, suggesting that this substrate is favored by the enzyme under such conditions. RNAi targeting PcIDS1 revealed the participation of this enzyme in the de novo synthesis of defensive monoterpenoids in the beetle larvae. As an FDP synthase, PcIDS1 could be associated with the formation of sesquiterpenes, such as juvenile hormones. Detection of Co , or Mg 2+ in the beetle larvae suggests flux control into C 10 vs. C 15 isoprenoids could be accomplished by these ions in vivo. The dependence of product chain length of scIDSs on metal cofactor identity introduces an additional regulation for these branch point enzymes of terpene metabolism.cobalt | kinetic | prenyltransferase | secretions | silencing

Frontalin pheromone biosynthesis in the mountain pine beetle, Dendroctonus ponderosae , and the role of isoprenyl diphosphate synthases

“…Dual-function GPPS/FPPS enzymes have been reported previously in other insects (12,29,30). In the horseradish leaf beetle, P. cochleariae, the GPP/FPP product ratio is affected by the divalent metal cofactor used in the assay (12). Using site-directed mutagenesis, a previous study has shown that two residues (indicated in bold) preceding the first aspartate-rich motif of the IDS from the aphid Acyrthosiphon pisum (ApisGPPS/FPPS, YQLVLDDIMD, at positions 194-203 of the alignment in Fig.…”

“…In I. pini, the IPPI transcript is up-regulated by both juvenile hormone III and feeding in adults for pheromone biosynthesis (27,28). Dual-function GPPS/FPPS enzymes have been reported previously in other insects (12,29,30). In the horseradish leaf beetle, P. cochleariae, the GPP/FPP product ratio is affected by the divalent metal cofactor used in the assay (12).…”

“…S4). For comparison, the human FPPS, the cotton boll weevil FPPS, and the horseradish leaf beetle GPPS/FPPS are dimers (11,12,16); human GGPPS is an octamer (17); and bovine GGPPS is a multimer of four to five units (18).…”

“…DponGPPS/FPPS protein was 99% identical to a putative FPPS from Dendroctonus jeffreyi (13) and shared 75%, 70%, 62%, 62%, 50%, and 49% amino acid identity with the FPPSs from A. grandis (11), I. pini (13), Tribolium castaneum (GenBank accession no. XP_972537), P. cochleariae (12), Choristoneura fumiferana (19), and Drosophila melanogaster (GenBank accession no. CAA08919), respectively (Fig.…”

“…A few IDSs have been characterized in Coleoptera, including an FPPS from the cotton boll weevil (Anthonomus grandis) (11), a GPPS/FPPS from the horseradish leaf beetle (Phaedon cochleariae) (12), and a unique GPPS from the pine engraver beetle (Ips pini) that also produces myrcene (13,14). To assess their importance in pheromone biosynthesis, we identified and functionally characterized two IDSs in MPB.…”