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Farnesyl diphosphate synthase (FPPS) catalyzes the condensation of the non-allylic diphosphate, isopentenyl diphosphate (IPP; C 5 ), with the allylic diphosphate primer dimethylallyl diphosphate (DMAPP; C 5 ) to generate the C 15 prenyl chain (FPP) used for protein prenylation as well as sterol and terpene biosynthesis. Here, we designed and prepared a series of pyridinium bisphosphonate (PyrBP) compounds, with the aim of selectively inhibiting FPPS of the lepidopteran insect order. FPPSs of Drosophila melanogaster and the spruce budworm, Choristoneura fumiferana , were inhibited by several PyrBPs, and as hypothesized, larger bisphosphonates were more selective for the lepidopteran protein and completely inactive towards dipteran and vertebrate FPPSs. Cell growth of a D. melanogaster cell line was adversely affected by exposure to PyrPBs that were strongly inhibitory to insect FPPS, although their effect was less pronounced than that observed upon exposure to the electron transport disrupter, chlorfenapyr. To assess the impact of PyrBPs on lepidopteran insect growth and development, we performed feeding and topical studies, using the tobacco hornworm, Manduca sexta , as our insect model. The free acid form of a PyrBP and a known bisphosphonate inhibitor of vertebrate FPPS, alendronate, had little to no effect on larval M. sexta ; however, the topical application of more lipophilic ester PyrBPs caused decreased growth, incomplete larval molting, cuticle darkening at the site of application, and for those insects that survived, the formation of larval–pupal hybrids. To gain a better understanding of the structural differences that produce selective lepidopteran FPPS inhibition, homology models of C. fumiferana and D. melanogaster FPPS ( Cf FPPS2, and Dm FPPS) were prepared. Docking of substrates and PyrBPs demonstrates that differences at the −3 and −4 positions relative to the first aspartate rich motif (FARM) are important factors in the ability of the lepidopteran enzyme to produce homologous isoprenoid structure and to be selectively inhibited by larger PyrBPs.

Farnesyl diphosphate synthase (FPPS) is an enzyme from the class of short chain ( E)-prenyltransferases that catalyzes the condensation of two molecules of isopentenyl diphosphate (IPP, C5) with dimethylallyl diphosphate (DMAPP, C5) to generate the C15 product FPP. In insects, FPPS plays a key role in the biosynthesis of the morphogenetic and gonadotropic “juvenile hormone” (JH). Lepidopteran genomes encode two very distinct FPPS paralogs , one of which (“type-II”) is expressed almost exclusively in the JH-producing glands, the corpora allata . This paralog has been hypothesized to display structural features that enable the binding of the bulkier precursors required for the biosynthesis of lepidopteran ethyl-branched JHs. Here, we report on the first crystal structures of an insect FPPS solved to date. Apo, ligand-bound, and inhibitor-bound structures of type-II FPPS (FPPS2) from the spruce budworm , Choristoneura fumiferana (Order: Lepidoptera), were obtained. Comparison of apo and inhibitor-bound enzymes revealed differences in both inhibitor binding and structural plasticity of CfFPPS2 compared to other FPPSs. Our data showed that IPP is not essential to the closure of the C-terminal tail . Ortho-substituted pyridinium bisphosphonates, previously shown to inhibit CfFPPS2, bound to the allylic site, as predicted; however, their alkyl groups were oriented towards the homoallylic binding site, with the bulkier propyl-substituted inhibitor penetrating deeply into the IPP binding pocket. The current study sheds light on the structural basis of substrate specificity of type-II FPPS of the spruce budworm. Through a comparison with other inhibitor-bound FPPSs, we propose several approaches to improve inhibitor selectivity and potency.

Isopentenyl diphosphate isomerase (IPPI) of the spruce budworm, Choristoneura fumiferana, and of the tobacco hornworm, Manduca sexta, was cloned and its catalytic properties assessed. In the presence of Mg(2+) or Mn(2+), the recombinant protein from C. fumiferana (CfIPPI) efficiently isomerized IPP to dimethylallyl diphosphate (DMAPP). While C. fumiferana IPPI transcript levels were evenly distributed in a wide variety of tissues, they were highly abundant in the corpora allata. Because IPPI plays an alternate role in lepidopteran juvenile hormone (JH) biosynthesis by catalyzing the isomerization of the homologous substrate, homoisopentenyl diphosphate (HIPP), the ability of CfIPPI to convert HIPP to homodimethylallyl diphosphate (HDMAPP) was also studied. As expected, HIPP isomerization was efficient and the formation of HDMAPP occurred, but the regiospecificity of the reaction was lower than previously found in M. sexta corpora allata homogenates and with purified Bombyx mori IPPI. Differences in inhibitory potency for several alkylated ammonium diphosphates and higher homologs of DMAPP were noted between CfIPPI and a vertebrate IPPI, suggesting that the lepidopteran enzyme has a larger active site cavity. To determine the structural factors responsible for homologous substrate coupling, site directed mutagenesis of several residues identified through sequence alignment and homology modeling analysis was performed. The results suggest that unlike other IPPIs, W216 (C. fumiferana numbering) works in concert with a tyrosine residue (Y105) to allow binding of larger substrates and to stabilize the high-energy intermediate formed during substrate isomerization.

Two forms of farnesyl diphosphate synthase (FPPS) from the spruce budworm, Choristoneura fumiferana, and one from the armyworm Pseudaletia unipuncta, have been cloned and their catalytic properties assessed. The type-2 FPPS of C. fumiferana (CfFPPS2) was efficient in the prenyl coupling of DMAPP or GPP with [14C]IPP, producing FPP as its final product; however, type-1 FPPSs (CfFPPS1, PuFPPS1, as well as Agrotis ipsilon FPPS1) were essentially inactive. A variety of purification methods was employed to purify the type-1 enzymes. Under mild chromatographic conditions, the isolated type-1 enzyme showed modest activity, but was apparently contaminated with endogenous prenyltransferase derived from the Escherichia coli host cells. Similarly, unpurified extracts of PuFPPS1 expressed in an E. coli FPPS-null mutant, had low FPPS activity. When equimolar amounts of homogenous CfFPPS1 and CfFPP2 were combined, a sharp synergistic enhancement of activity was observed, and the coupling of several homologous substrates, which are precursors to ethyl-branched JHs, was enhanced. Association between CfFPPS1 and CfFPPS2 was confirmed by both protein interaction chromatography and competitive ELISA. These data suggest that type-1 and type-2 FPPSs can form a heteromer, which may play a role in sesquiterpene biosynthesis, such as JH homologue formation, in moths.

Geranylgeranyl diphosphate synthase (GGPPS) catalyzes the condensation of the non-allylic diphosphate, isopentenyl diphosphate (IPP; C5), with allylic diphosphates to generate the C20 prenyl chain (GGPP) used for protein prenylation and diterpenoid biosynthesis. Here, we cloned the cDNA of a GGPPS from the spruce budworm, Choristoneura fumiferana, and characterized the corresponding recombinant protein (rCfGGPPS). As shown for other type-III GGPPSs, rCfGGPPS preferred farnesyl diphosphate (FPP; C15) over other allylic substrates for coupling with IPP. Unexpectedly, rCfGGPPS displayed inhibition by its FPP substrate at low IPP concentration, suggesting the existence of a mechanism that may regulate intracellular FPP pools. rCfGGPPS was also inhibited by its product, GGPP, in a competitive manner with respect to FPP, as reported for human and bovine brain GGPPSs. A homology model of CfGGPPS was prepared and compared to human and yeast GGPPSs. Consistent with its enzymological properties, CfGGPPS displayed a larger active site cavity that can accommodate the binding of FPP and GGPP in the region normally occupied by IPP and the allylic isoprenoid tail, and the binding of GGPP in an alternate orientation seen for GGPP binding to the human protein. To begin exploring the role of CfGGPPS in protein prenylation, its transcripts were quantified by qPCR in whole insects, along with those of other genes involved in this pathway. CfGGPPS was expressed throughout insect development and the abundance of its transcripts covaried with that of other prenylation-related genes. Our qPCR results suggest that geranylgeranylation is the predominant form of prenylation in whole C. fumiferana.

Chitin deacetylase (CDA) catalyzes the conversion of chitin into chitosan, thereby modifying the physical properties of insect cuticles and peritrophic matrices. A lepidopteran chitin deacetylase gene (CfCDA2) was cloned from the spruce budworm, Choristoneura fumiferana, and found to generate two alternatively spliced transcripts, CfCDA2a and CfCDA2b. Transcriptional analysis using isoform-specific RT-PCR primers indicated that both isoforms were upregulated during the molt. Interestingly, CfCDA2b transcripts were most abundant in the head during the molting stage while those of CfCDA2a were predominant in the epidermis during the feeding period. Injection of CfCDA2-specific dsRNA into C. fumiferana larvae or pre-pupae induced both abnormal phenotypes and high mortality, which resulted from an inability to shed the old cuticle. These results suggest that CfCDA2 plays an important role in the molting process, and that the two alternatively spliced transcripts have different functions during insect development. This is the first detailed characterization of lepidopteran chitin deacetylase gene.

We tested the hypothesis that shifts in the proportions of the various juvenile hormones (JHs) and precursor acids released by lepidopteran corpora allata (CA) are associated with changes in the activity of enzymes involved in the production of propionate from isoleucine. Measurements of enzyme activity were made on CA of adult Pseudaletia unipuncta , using the assay developed by Brindle et al [ Arch. Insect Biochem. Physiol ., 19, 1 (1992)]. Female and male CA of P. unipuncta release the three principal forms of JH and JH acid (JHA), respectively, in proportions that vary according to age and total hormonal output. In addition, female CA incorporate isoleucine-derived propionate into JH I and JH II in a stoichiometric fashion. The branched-chain amino acid (BCAA) transaminase, an enzyme that catalyzes the first step in the conversion of isoleucine into propionate, was 5–25 times more active in homogenates of male CA than in those of females, and its activity increased as a function of age in males but not in females. Similar trends were observed for the accumulation of metabolites produced in later steps of this conversion. These observations support the hypothesis that the age-related increase in the biosynthesis of JHA I by male CA is associated with a rise in the production of propionate, whereas limited availability of propionate in female glands leads to an age-dependent decrease in the proportion of JH I released. These results also imply the existence of a mechanism regulating propionate production in lepidopteran CA. The regulatory factor, however, is not Manduca allatostatin as it had no detectable effect on isoleucine metabolism by either intact male or female CA. Lastly, changing the concentration of acetate and propionate in the CA incubation medium had a greater impact on the proportions of JH I and JH II released than on the proportion of JH III.

In addition to providing lipid chains for protein prenylation, short-chain isoprenyl diphosphate synthases (scIPPSs) play a pivotal role in the biosynthesis of numerous mevalonate pathway end-products, including insect juvenile hormone and terpenoid pheromones. For this reason, they are being considered as targets for pesticide development. Recently, we characterized an aphid scIPPS displaying dual geranyl diphosphate (GPP; C 10 )/farnesyl diphosphate (FPP; C 15 ) synthase activity in vitro . To identify the mechanism(s) responsible for this dual activity, we assessed the product selectivity of aphid scIPPSs bearing mutations at Gln107 and/or Leu110, the fourth and first residue upstream from the “first aspartate-rich motif” (FARM), respectively. All but one resulted in significant changes in product chain-length selectivity, effectively increasing the production of either GPP (Q107E, L110W) or FPP (Q107F, Q107F–L110A); the other mutation (L110A) abolished activity. Although some of these effects could be attributed to changes in steric hindrance within the catalytic cavity, molecular dynamics simulations identified other contributing factors, including residue-ligand Van der Waals interactions and the formation of hydrogen bonds or salt bridges between Gln107 and other residues across the catalytic cavity, which constitutes a novel product chain-length determination mechanism for scIPPSs. Thus the aphid enzyme apparently evolved to maintain the capacity to produce both GPP and FPP through a balance between these mechanisms.

Farnesyl diphosphate synthase (FPPS) of the dipteran Drosophila melanogaster has been cloned and its catalytic properties have been assessed. Analysis of the D. melanogaster genome and of ESTs indicates that FPPS is a single copy gene that produces two transcripts, which differ only by 5' extension. The cDNA of shorter and longer D. melanogaster FPPSs (DmFPPS-1a and DmFPPS-1b, respectively) were each subcloned into pET28a and expressed as an N-His6 fusion protein in BL21 E. coli cells. The DmFPPSs similarly catalyzed the coupling of the allylic substrates, GPP and DMAPP, with IPP, producing FPP as product. The longer protein was further characterized. The enzyme required divalent metal for activity, and was activated by 0.1% Triton X-100. Higher detergent concentration and the addition of glycerol, conditions that activate certain insect FPPSs, inhibited prenyl coupling by DmFPPS-1b. Although DmFPPS-1b does not efficiently couple homologous GPP compounds, homodimethylallyl diphosphate (HDMAPP), which is precursor to all homologous JH structures, was a reactive substrate.

A 15-residue neuropeptide, Manduca sexta allatostatin (Mas-AST), strongly inhibits juvenile hormone (JH) biosynthesis in vitro by corpora allata (CA) from Manduca fifth-stadium larvae and adult females as well as Helicoverpa zea adult females (Kramer et al. , 1991. Proc. Natl. Acad. Sci (USA) 88, 9458–9462). In contrast, this study found that 1.0 μM Mas-AST has no JH biosynthesis inhibitory activity in Pseudaletia unipuncta sixth instar larvae or newly-emerged (day 0) adults but inhibited CA of 5-day-old adult females by 60%. From a P. unipuncta brain cDNA library, was isolated a cDNA that encodes a 125 amino acid polypeptide containing the Mas-AST sequence. Within the precursor, Mas-AST is situated at the car☐y terminus and is flanked by different dibasic proteolytic cleavage signals. The Pseudaletia gene specifying the Mas-AST peptide is present as a single copy per haploid genome. Expression of this gene was low in Pseudaletia sixth instar larvae, prepupae and early pupae but was relatively high in late pupae, and day 1 and 3 adults of both sexes. In day 5 adults, the relative transcript level appears to be maintained in females but declines in males. This pattern of Mas-AST expression does not correlate well with the profile of JH biosynthesis in Pseudaletia , which increases during the first 5 days of adult life, suggesting additional or alternative functions for this peptide.