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    • In general FPSs require Mg or Mn as a cofactor

    2021-11-30

    In general, FPSs require Mg2+ or Mn2+ as a cofactor for GPP and FPP synthesis. Co2+ also acts as a cofactor in EuFPSs, as in the case for PcFPS and Abies grandis FPS [29], [44]. Co2+ enhanced the activity of EuFPS1 more than the activity of EuFPS2: Mg2+, Mn2+ and Co2+ had approximately the same effect on the activation of EuFPS1, whereas Co2+ was less effective than Mg2+ and Mn2+ toward EuFPS2 (Fig. 4). Both EuFPSs likely utilize Mg2+ and Mn2+ as the main cofactors due to the toxicity of Co2+ to plant cells [45]. Indeed, although various enzymes require metal ion for activity, and a plant Co2+ transporter has been identified [46], [47], only a handful of plant enzymes utilize Co2+ as a cofactor (Table S1). Therefore, Co2+ is probably not used as a cofactor by EuFPSs in vivo whereas Mg2+ or Mn2+ are required as the main cofactor for EuFPSs in vivo. The two most notable differences between the EuFPSs were their specific activities and product preferences. Under most reaction conditions used in this study, EuFPS2 showed over twice the specific activity of EuFPS1. EuFPS1 elongated DMAPP to FPP and little GPP was accumulated as an intermediate product under most reaction conditions. In contrast, EuFPS2 showed essentially unchanged GPP synthetic activity in the presence of DMAPP, suggesting that EuFPS1 reaction is strictly regulated to synthesize FPP, and that FPP synthesis from DMAPP mediated by EuFPS2 is not always a sequential condensation reaction. The allylic substrate- and ion-dependent end product preferences of EuFPS2 were also observed with PcFPS [29], suggesting common features contributing to the enzymatic characteristics of EuFPS2 and PcFPS. There were no remarkable differences among the amino i am g similarities of the EuFPSs and PcFPS except for the increased length of the N-terminal sequence of PcFPS (Table S2). However, this long N-terminal sequence does not affect PcFPS function because PcFPS contains the cleavage site for a mitochondrial targeting sequence, RxxS motif, at Arg67-Ser70, and the N-terminal truncated form of PcFPS shows ion-dependent product preferences [29]. Therefore, another factor(s) might affect the activities of FPSs. It is noteworthy that FPSs undergo an open-to-closed structural transition during catalysis, and substrate binding to FPSs triggers this transition, owing to the rearrangement of the catalytic loops and the helices α4 and α6 (Fig. 5A and B). Although the homology models of the open and closed forms of EuFPS1, EuFPS2 and PcFPS are nearly identical (Fig. S9), slight but significant structural differences are observed in the open and closed models of these three enzymes. The presence or absence of hydrogen-bond between Tyr88 and Glu154 in EuFPS1 likely affects structural dynamics during the open-to-closed transition (Fig. 5). The hydrogen-bond is not observed in EuFPS2 and PcFPS, which raise the possibility that such structural features are responsible for the differences noted in the enzymatic characteristics between EuFPS1 and EuFPS2 or PcFPS. Novel insect FPSs were recently identified and characterized [48]. Depending on the allylic substrate, one novel FPS, PsIDS3 (Phyllotreta striolata isoprene diphosphate synthase 3), synthesized (Z)-GPP, (Z,Z)-FPP or (Z,E)-FPP. Similar to other FPSs, PsIDS3 contains highly conserved motifs but produces different reaction products, suggesting that there are novel determinant residues for the synthesis of FPP isoforms. Tyr88/Phe95 in EuFPSs are believed to form a floor in the active center of eukaryotic enzymes and affect CLD, and structural modeling suggests that these residues may affect function and structural stability. Other amino acids might also contribute to FPS activity and structures and thus a comparison of the amino acid sequences and catalytic properties of FPSs might identify new determinants of FPP catalytic activity and structural stability. In conclusion, we identified two highly homologous farnesyl diphosphate synthases in Eucommia and their distinct enzymatic properties including substrate preferences, substrate inhibition, and metal-ion-depended end product. Our results suggest that FPS has novel key amino acid residue(s) regulating the biosynthesis of short chain polyprenoids.