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  • Most studies to date have focused on the physiological


    Most studies to date have focused on the physiological functions of FPS in plants. As mentioned above, numerous factors affect FPS expression and FPS function. However, FPS enzymatic assays require radiolabeled substrate and thus detailed enzymatic characterizations of FPS, including substrate specificity, ion dependency, and product preferences need further characterization. Human FPS indeed showed the substrate inhibition, and it was still unclear. Recently, a high-throughput and high-sensitivity method was established for analyzing FPS reaction products using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) [28]. This method allows elucidation of the ion dependencies, the product preference, and substrate inhibition of insect FPS [29]. The method could provide us more information about the detail reaction mechanisms of FPS. Since plants have redundant FPSs, a high-throughput analysis using LC-MS/MS should help identify enzymatic differences between the redundant FPSs. In this study, we focused on characterizing the FPSs from Eucommia ulmoides (EuFPSs) for a following reason: two uncharacterized homologues of E. ulmoides might have different enzymatic properties and function in Eucommia in spite of their high amino selective serotonin reuptake inhibitors sequence similarities. The characteristics of EuFPSs are discussed in this study, as are elucidations for the functional regulation of FPS.
    Materials and methods
    Discussion FPP is an essential compound for the biosynthesis of most linear isoprenoids, sterols and sesquiterpenes, and for protein farnesylation. In E. ulmoides, FPP is believed to be utilized as an allylic substrate in long chain trans-polyisoprene biosynthesis, and thus there is an interest in elucidating the mechanism of FPP biosynthesis and in characterizing EuFPSs. Decades of research on FPP synthesis have resulted in the identification and characterization of many FPSs from bacteria, insects, plants, and mammals. Unfortunately, classical approaches using radiolabeled chemicals provided limited information and therefore there are no overviews of the characteristics of FPSs. Recently, FPS from Phaedon cochleariae was characterized using LC-MS/MS and novel FPS mechanisms of reaction were determined [29]. Instrumental analysis is thus displacing classical approaches and provide a powerful tool for elucidating of FPS functions. Expanding on previous report [28], here we developed a UPLC-MS/MS method enabling both PDA detection of short-chain polyisoprenyl diphosphates and more rapid analysis with high sensitivity than conventional HPLC analysis. In addition to biological analyses, a high-throughput approach used in this study provided significant information regarding distinct enzymatic properties arising from amino acid differences in highly conserved motifs. The recombinant E. coli produced soluble EuFPSs that synthesized FPP as the major product from DMAPP and GPP. In addition, the EuFPSs utilized FPP as an allylic substrate and synthesized GGPP, albeit less effectively than FPP, demonstrating that EuFPSs are bifunctional prenyltransferases with major FPS activity and a faint GGPP synthase activity. The two EuFPSs exhibit high amino acid sequence similarity, yet their properties, such as kinetics and metal ion dependencies, were different, and their specific activities were particularly different. This agrees with the results of complementation analyses of the S. cerevisiae Δfps mutant which showed EuFPS2 weakly complemented fps-deficiency on glucose plate using a little endogenous galactose [38]. However, the activities of the EuFPSs were inhibited by IPP and DMAPP in a similar manner, suggesting that DMAPP (rather than IPP) might preferentially bind to EuFPS2 and inhibit FPP synthesis. Despite the identification and characterization of various FPSs and their inhibitors, there are only a few reports of substrate inhibition of FPS; for example, the activities of human FPS and PcFPS are inhibited by IPP [29], [39], [40]. The activities of the EuFPSs were also inhibited by IPP (>100 μM; 1:1 M ratio), suggesting that the active site near the first DDxxD motif (which contributes to allylic substrate binding) is occupied by IPP. A previous study showed that two IPP molecules bind to the avian FPS monomer [41], [42], suggesting that IPP can bind both to the donor and allylic sites. Thus, the vicinity of the FPS active site apparently has sufficient space and/or flexibility to support the binding of both substrates. This speculation is supported by the finding that allylic substrates could access the second DDxxD motif (which ordinarily contributes to the binding of the donor substrate IPP) [36], [43]. However, this theory is confounded by the discovery that each subunit of avian FPS forms a 1:1 complex with DMAPP, GPP or FPP [41], indicating that allylic substrates are likely unable to bind to the donor substrate binding site. Conversely, a high concentration of GPP resulted in substrate inhibition in PcFPS [29], suggesting that the first DDxxD motif has high affinity for GPP as shown by the low Km value for GPP, whereas the relative binding affinity to the second DDxxD motif is quite low. Interestingly, avian FPS and PcFPS show low binding affinity for DMAPP, whereas the EuFPSs enables the binding of DMAPP to both the donor and allylic sites, resulting in both FPP synthesis and substrate inhibition. These results suggest that differences in the amino acid sequence and/or tertiary structure around the active site, rather than differences in affinities of the DDxxD motifs for the allylic substrate(s), affect the properties of FPSs.