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

Abstract

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-β and MhiePF/GGT-α) were identified in the leaf beetle, Monolepta hieroglyphica. Phylogenetic analysis revealed that MhieFPPS1, MhieFPPS2, MhiePFT-β 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-β and MhiePF/GGT-α) lack the aspartate-rich motif. Docking results indicated that binding site of MhiePFT-β 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-β operated via Zn2+-dependent substrate binding, while MhiePF/GGT-α stabilized the β-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.