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  • The deduced amino acid sequences of all

    2022-06-29

    The deduced amino AZD8055 sequences of all scIPPSs display high similarity, and sequence alignments reveal seven conserved regions [2]. Regions II and VI contain the substrate-binding regions, with two characteristic aspartate-rich motifs (DDx(xx)xD; where x represents any amino acid residue). Region II, which includes the first aspartate-rich motif (FARM), is designated as the chain-length determination region, whereas, the second aspartate-rich motif (SARM) in region VI is responsible for IPP binding [3]. In insects, FPPS is involved in the biosynthetic pathway of the sesquiterpenoid juvenile hormone, which has important morphogenetic and gonadotropic functions [4]. In many aphid species, FPPS is also predicted to play a key role in the biosynthetic pathway of the alarm pheromone E-β-farnesene [5]. Whereas FPPS and GGPPS occur nearly ubiquitously in plants, animals, fungi and bacteria, GPPS appears to have a more limited distribution in nature, having been identified almost exclusively in essential oil (monoterpene)-producing plants. However, monoterpenes are also important natural products in some insects, serving as semiochemicals. Recently, evidence for the existence of GPPS activity in insects was provided for scolytid [6] and chrysomelid beetles [7]. In aphids, the presence of a GPPS is also expected inasmuch as the cyclopentanoids nepetalactone and nepetalactol, which are derived from GPP, are essential components of the sex pheromone [8]. In spite of the presumed importance of scIPPSs in insect metabolism, a limited number of insect IPPS sequences have been reported to date, and characterization of the recombinant proteins has only recently begun [6], [9], [10], [11], [12], [13]. In this paper, we report on the cloning of three scIPPS cDNAs from M. persicae (two long forms, MpIPPS1-L and MpIPPS2-L and a short form, MpIPPS1-S), on their heterologous expression and on the characterization of one of these isoforms, including the identification of its principal in vitro products, prediction of its 3D-structure, and analysis of the generated 3D models. Although MpIPPS1 isoforms were initially predicted to be FPPSs, based on their amino acid sequences, in vitro assays revealed a dual GPP/FPP synthase activity, generating both GPP and FPP.
    Materials and methods
    Results
    Discussion Although prenyltansferases capable of producing both FPP and GGPP were reported earlier for two archeas [23] and more recently for the protozoan parasite Toxoplasma gondii[24], our study conducted on the aphid M. persicae led to the identification of the first prenyltransferase that can generate both GPP and FPP. In general FPPSs have been reported to yield about 3 times or more FPP than GPP [13], [25], [26], [27]. Unexpectedly, rMpIPPS1-S yielded a GPP:FPP ratio varying between ∼50:50 and ∼70:30, depending on DMAPP concentration, while the dipteran rDmFPPS produced about 90% FPP. The latter observation suggests that rMpIPPS1 plays a predominant role in GPP formation, and that its function is not limited to FPP production, as originally hypothesized. Nervertheless, unlike other GPPSs known, the rMpIPPS1-S was shown here to be capable of using GPP as allylic co-substrate to generate significant amounts of FPP. Under our in vitro assay conditions, the GPP/FPP ratio was dependent on DMAPP concentration. At higher DMAPP concentration, the rMpIPPS1-S produced increasing amounts of GPP. This observation is in agreement with earlier reports indicating that the product profile of scIPPSs may be, to a certain degree, modulated by substrate concentration [28]. In vivo, the DMAPP concentration could be under the control of IPP isomerase, which catalyzes the isomerization of IPP to DMAPP, or could be regulated by substrate compartmentalization. Some of the positions identified here (Phe112/Tyr106, Phe113/Gln107, Ala116/Leu110) have been shown to be involved in product chain-length selectivity in the avian enzyme, using site-directed mutagenesis [29], [30].