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  • To examine the biochemical characteristics of FPPS in

    2022-01-26

    To examine the biochemical characteristics of FPPS in Drosophila, DmFPPS has been functionally expressed in E. coli. The protein displays properties similar to mammalian FPPS, with some notable differences, including the ability to efficiently couple the homologous substrate, homodimethylallyl diphosphate (HDMAPP).
    Materials and methods
    Results
    Discussion FPPS plays a central role in isoprenoid biosynthesis. In D. melanogaster, FPPS is encoded by a single copy gene, the function of which could be inferred from recent yeast complementation studies (Cusson et al., 2006), and from its sequence similarity to other known prenyltransferases. However, the precise enzymatic properties of this enzyme were not known. DmFPPS was heterologously expressed in E. coli, and was subsequently purified by Co2+-based IMAC. The activity of the longer and shorter versions of this protein was found to be essentially identical to one another and to the corresponding recombinant protein that lacks the N-histidine tag (Table 4). DmFPPS showed many of the characteristics considered typical of prenyltransferases, including a requirement of divalent cation (Mg2+ or Mn2+) and an ability to couple both DMAPP and GPP to IPP. Given that the Lepidoptera are known to produce homologous JH structures and that this capability is believed to be associated, at least in part, with the substrate specificity of FPPS of the corpora allata (the endocrine glands that produce JH, Sen et al., 2006), we were interested in determining whether DmFPPS had substrate specificity and enzymological properties similar to those of lepidopteran prenyltransferase. The latter has only recently been assessed using recombinant CORM-3 (Sen et al., 2007a), while two FPPSs (FPPS I and FPPS II) have been purified from Bombyx mori (Koyama et al., 1985) and another one has been isolated from Manduca sexta (Sen and Sperry, 2002). Also, the properties of larval and adult M. sexta prenyltransferase, derived from corpora allata homogenates, have been examined (Sen et al., 2007b). Using these studies as a basis for comparison, we first measured the effect of several additives on DmFPPS-1b activity, including the non-ionic detergent Triton X-100 and the protein stabilizer glycerol, both of which are known to activate lepidopteran prenyltransferases. While up to 2% (w/v) Triton X-100 activates B. mori FPPS I (Koyama et al., 1985) and crude preparations of M. sexta prenyltransferase (Sen et al., 1996), we found that DmFPPS-1b was strongly inhibited at this concentration. Glycerol also inhibited DmFPPS-1b activity, indicating that this protein has properties that differ from those of lepidopteran FPPSs. Analogs of DMAPP and GPP that are precursors of lepidopteran JHs were tested for their ability to serve as substrates of DmFPPS-1b. GPP and its homologs followed the reactivity trend: GPP>iso-homo-GPP>homo-GPP>bishomo-GPP, suggesting that DmFPPS-1b, unlike the FPPS-II of B. mori (Koyama et al., 1985), lacks the ability to efficiently couple homologous substrates and does not discriminate against iso-homo-GPP, which is precursor to a JH structure found so far in only one lepidopteran species (Kou et al., 1995). In contrast, when HDMAPP (which is a homolog of DMAPP and is required for the biosynthesis of all ethyl-branched JHs) was utilized as allylic substrate, we observed that it was slightly preferred over DMAPP, in sharp contrast to vertebrate and plant FPPSs, which disfavor coupling of this homologous substrate (Nishino et al., 1972, Nishino et al., 1973; Ogura et al., 1968).
    Conclusion The results presented here provide the first functional characterization of a dipteran FPPS. DmFPPS displays properties that appear distinct from those of either conventional or lepidopteran FPPSs. The ability of DmFPPS-1b to couple certain homologous substrates is surprising, given that the residues that line its active site, unlike those of type-I lepidopteran FPPSs (Cusson et al., 2006), do not show notable substitutions relative to those of conventional eukaryotic FPPSs. In a phylogenetic tree that we reported earlier (Cusson et al., 2006), the dipteran, coleopteran and lepidopteran (type-I) FPPS branches were collapsed due to low bootstrap support for the observed branching order. Interestingly, however, all preliminary trees placed the dipteran FPPSs as most closely related to those of the Lepidoptera, a relationship that could prove to be correct should greater taxonomic sampling be achieved. Although the longer and shorter versions of DmFPPS appear to have identical enzymatic properties, it is not clear whether they have different roles in insect isoprenoid (including JH) metabolism. Further studies will be needed to determine whether DmFPPS compartmentalization serves a role in regulating isoprenoid flux, as occurs in plants.