Functional characterization of a geranylgeranyl diphosphate synthase in the leaf beetle Monolepta hieroglyphica.

Geranylgeranyl diphosphate synthase (GGPPS) as the short-chain prenyltransferases for catalyzing the formation of the acyclic precursor (E)-GGPP has been extensively investigated in mammals, plants, and microbes, but its functional plasticity is poorly understood in insect species. Here, a single GGPPS in leaf beetle Monolepta hieroglyphica, MhieGGPPS, was functionally investigated. Phylogenetic analysis showed that MhieGGPPS was clustered in one clade with homologs and had six conserved motifs. Molecular docking results indicated that binding sites of dimethylallyl diphosphate (DMAPP), (E)-geranyl pyrophosphate (GPP), and (E)-farnesyl pyrophosphate (FPP) were in the chain-length determination region of MhieGGPPS, respectively. In vitro, recombiant MhieGGPPS could catalyze the formation of (E)-geranylgeraniol against different combinations of substrates including isopentenyl pyrophosphate (IPP)/DMAPP, IPP/(E)-GPP, and IPP/(E)-FPP, suggesting that MhieGGPPS could not only use (E)-FPP but also (E)-GPP and DMAPP as the allylic cosubstrates. In kinetic analysis, the (E)-FPP was most tightly bound to MhieGGPPS than that of others. It was proposed that MhieGGPPS as a multifunctional enzyme is differentiated from the other GGPPSs in the animals and plants, which only accepted (E)-FPP as the allylic cosubstrate. These findings provide valuable insights into understanding the functional plasticity of GGPPS in M. hieroglyphica and the novel biosynthesis mechanism in the isoprenoid pathway.

Functional characterization of prenyltransferases involved in de novo synthesis of isoprenoids in the leaf beetle Monolepta hieroglyphica.

Prenyltransferases play a pivotal role in the isoprenoid biosynthesis and transfer in insects. In the current study, two classes of prenyltransferases (MhieFPPS1 and MhieFPPS2, MhiePFT-ß and MhiePF/GGT-α) were identified in the leaf beetle, Monolepta hieroglyphica. Phylogenetic analysis revealed that MhieFPPS1, MhieFPPS2, MhiePFT-ß and MhiePF/GGT-α were clustered in one clade with homologous in insects. Moreover, MhieFPPS2 lacked one aspartate-rich motif SARM. Molecular docking and kinetic analysis indicated that the (E)-GPP displayed higher affinity with MhieFPPS1 compared to DMAPP within the binding pocket containing metal binding sites (MG). The other class of prenyltransferases (MhiePFT-ß and MhiePF/GGT-α) lack the aspartate-rich motif. Docking results indicated that binding site of MhiePFT-ß involved divalent metal ions (Zn) and bound farnesyl or geranylgeranyl. In vitro, only recombiant MhieFPPS1 could catalyze the formation of (E)-farnesol against different combination of substrates, including IPP/DMAPP and IPP/(E)-GPP, highlighting the importance of SARM for enzyme activities. Kinetic analysis further indicated that MhiePFT-ß operated via Zn2+-dependent substrate binding, while MhiePF/GGT-α stabilized the ß-subunit during catalytic reaction. These findings contribute to a valuable insight in to understanding of the mechanisms involved in the biosynthesis and delivery of isoprenoid products in beetles.