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    • A species specific diverse number of FPPS

    2022-01-21

    A species-specific diverse number of FPPS genes are present in insect genomes (Zhang and Li, 2008, Cusson et al., 2006). The genome of the yellow fever mosquito Aedes aegypti contains a single FPPS gene (AaFPPS) that is highly expressed in the CA (Nouzova et al., 2011). FPPS catalytic activity in the nanomolar range was described in mosquito CA extracts, with changes in activity that were concurrent with increases and decreases of JH synthesis (Rivera-Perez et al., 2014). Given the critical role of FPP as a JH precursor, we set out to establish the functional characterization of AaFPPS, with a special emphasis on the role of metal ions on product specificity. The bases of our studies consisted of a series of in vitro assays testing a diversity of allylic substrates (IPP, DMAPP and GPP) and divalent cations (Co2+, Mg2+). Our results revealed that AaFPPS synthesizes farnesyl diphosphate from IPP and DMAPP, as well as from GPP and IPP. The enzyme requires metal cofactors for its activity and produces preferentially FPP in the presence of Mg2+, revealing a switch of product specificity toward GPP in the presence of Co2+. In addition, we showed that in the presence of an equimolar mix of both metal cofactors, AaFPPS synthesized a 1:1 proportion of both products, but in significant lower concentrations compared to reactions who only contained one cofactor, suggesting a potential impeding isoquercitrin mg of both ions for the metal binding motif (DDXXD).
    Material and methods
    Results
    Discussion Short-chain isoprenyl diphosphate synthases are a class of prenyltransferases that includes geranyl diphosphate synthase (GPPS), farnesyl diphosphate synthase (FPPS) and geranylgeranyl diphosphate synthase (GGPPS), which synthesize geranyl diphosphate (GPP) (C10), FPP (C15), and GGPP (C20), respectively. While FPPS and GGPPS are ubiquitous in nature, GPPS is largely restricted to plant species (Sommer et al., 1995, Burke et al., 1999), but has been also described in insects (Gilg et al., 2005). Isoprenyl diphosphate synthases catalyze the head-to-tail condensation of DMAPP with two molecules of IPP to sequentially generate GPP, FPP and GGPP (Kellogg and Poulter, 1997). The specificity and ratio of products generated depends on the interactions established in the catalytic pocket between allylic substrates (IPP, DMAPP, GPP and FPP) and divalent metal cofactors (Co2+, Mg2+ or Mn2+), with the reaction proceeding and terminating precisely at a specific carbon length (10, 15 or 20) according to the enzyme's product chain length specificity (Kellogg and Poulter, 1997). There are several examples of insect diphosphate synthases displaying “catalytic promiscuity”, and changing the type and ratio of products released (GPP or FPP) depending on allylic substrate concentrations (Sen et al., 2007a, Vandermoten et al., 2008) or the presence of different metal cofactors (Sen et al., 2007b, Frick et al., 2013). In insects, FPPSs are typically present as a single-copy gene, with exceptions in Hymenoptera, Homoptera and Lepidoptera (Vandermoten et al., 2009). In the Lepidoptera, FPPSs have long been suspected of exhibiting structural features allowing them to accommodate the bulkier homologous substrates and products used as precursors of ethyl-branched JHs (Cusson et al., 2013). We set out to establish the molecular bases of substrate specificity for AaFPPS, as well as the role of substrate concentration and metal ions on its catalytic specificity. Sequence analysis and homology modeling indicated that AaFPPS is a homodimeric protein, with each subunit containing a single site for chain elongation. AaFPPS showed a conserved alpha structure with the avian FPPS (Fig. 2A), with a catalytic pocket located in a central cavity formed by a bundle of 10 α-helices. The two critical aspartate rich sequences (DDXXD) were positioned on opposite sides of the cavity, and two hydrophobic amino acids (V178 and F179) adjacent to the first aspartate rich motif were sitting at the bottom of the pocket. These two aromatic amino acids would limit the access of long chain substrates to the hydrophobic pocket (Wang and Ohnuma, 1999, Wang and Ohnuma, 2000), defining structurally AaFPPS as a Class I short-chain FPPS. The dimension of the cavity suggests that there is enough space to incorporate GPP into the binding pocket and elongate it to FPP; this assumption was corroborated in our in vitro assays, where recombinant AaFPPS under different conditions produced GPP, FPP and even traces of GGPP.