46619 br Declarations of interest br
Declarations of interest
Founding information This work was partially supported by the New Energy and Industrial Technology Development Organization (NEDO) Japan [grant number P03043]; and Grants-in-Aid for Scientific Research [grant number 16H00738 to H.M.] from the Japan Society for the Promotion of Science (JSPS).
Author contributions HK and NS designed the research. HK produced, purified, and determined the properties of the recombinant EuFPSs. HK and YK performed tetrad analyses of S. cerevisiae. HK and ST established the analytical method for the EuFPS assay using UPLC-MS/MS. TY and HM constructed homology models of the EuFPSs and PcFPS and HK, TY and HM performed the structural comparisons. HK, YT, NS and HM analyzed all the data and wrote the manuscript. KF, YK and YN supervised the research.
Introduction The morphogenetic “juvenile hormone” (JH), found almost exclusively in insects, is an attractive target for the development of insecticides (Williams, 1967). Produced and released into the hemolymph by endocrine glands known as the corpora allata (CA) (Goodman and Cusson, 2012, Tobe and Stay, 1985), its main function is to regulate development and reproduction in insects (Jindra et al., 2013, Smykal et al., 2014). In immature insects, high levels of JH inhibit the early onset of 46619 during larval molts. In adult insects, JH promotes ovarian maturation, in addition to being involved in several other aspects of reproduction (Cusson and Palli, 2000). An artificially induced drop in JH titers prior to the last larval stadium typically leads to cessation of feeding followed by precocious metamorphosis, resulting in the formation of miniature adults that may not be able to survive or reproduce. Eight different structural forms of JH have been identified to date (Fig. 1). While all of them possess the sesquiterpenoid (C15) backbone, four of them, bearing one or more additional carbons, are specific to the Lepidoptera (JH and an isomeric form of it, 4-methyl-JH I, JH I and JH II). The first enzymatic steps leading to JH biosynthesis are those of the ubiquitous mevalonate pathway, whose end products are dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP), or the corresponding homologous structures. The construction of farnesyl diphosphate (FPP) or one of its ethyl-branched homologs is subsequently formed by the action of farnesyl diphosphate synthase (FPPS). Conversion of FPP to JH involves additional enzymatic steps specific to JH synthesis (Goodman and Cusson, 2012). FPPS catalyzes a reaction of carbon chain elongation dependent on the presence of divalent cations (usually Mg2+ and Mn2+), where the allylic isoprenoid diphosphate, DMAPP, serves as chain initiator in a condensation reaction involving the addition of two molecules of the non-allylic isoprenoid diphosphate, IPP, through electrophilic alkylation. While the genomes of most living organisms encode FPPS homologs, those of some insects, including the Lepidoptera, display taxon-specific structural features that could be taken advantage of in the development of selective inhibitors with insecticidal properties (Cusson et al., 2006, Sen et al., 1996, Vandermoten et al., 2009a, Vandermoten et al., 2009b). In most living organisms, FPPS is encoded by a single-copy gene. In the Lepidoptera, however, two very distinct paralogs have been identified: type-I (FPPS1) and type-II (FPPS2) FPPS (Cusson et al., 2006). Recently, a great diversification of FPPS paralogues, in addition to those corresponding to the orthologs of FPPS1 and FPPS2 of the Lepidoptera, was highlighted in the genus Papilio (Li et al., 2015). The roles of these “novel” FPPSs have yet to be identified. Tissue-specific transcriptional analyzes comparing transcript abundance of the type-I and type-II paralogs in Bombyx mori indicated that FPPS2 transcripts were largely confined to the CA, the exclusive site of JH biosynthesis, while those of FPPS1 were ubiquitously expressed. These data provide compelling evidence for the role of type-II FPPS in JH biosynthesis and suggest that this enzyme may be better suited than type-I FPPS for the production of FPP homologs containing ethyl branches (Cusson et al., 2006, Kinjoh et al., 2007). Interestingly, analysis of prenyltransferase activity in homogenates of CA from the lepidopteran Manduca sexta pointed to a preference of FPPS (presumed to be primarily FPPS2) for the C6 homolog of DMAPP (HDMAPP) over DMAPP, thus promoting the production of ethyl-branched JHs (Sen et al., 1996). These findings supported earlier studies showing that ethyl branches arise from the incorporation of propionyl-CoA into the mevalonate pathway, leading to the formation and substitution of the C6 homologs of IPP and DMAPP for the standard C5 isoprene units (Schooley et al., 1973).