In silico and in vitro analyses identified three amino acid residues critical to the catalysis of two aphid farnesyl diphosphate synthase.

Farnesyl diphosphate synthase (FPPS) plays an essential role in the isoprenoid biosynthetic pathway of microbes, plants and animals. In the present study, we first cloned two FPPSs from the bird cherry-oat aphid (RpFPPS1 and RpFPPS2), and activity assay by gas chromatography-mass spectrometry showed that both RpFPPS1 and RpFPPS2 were active in vitro. They were then subjected to homology modeling and molecular docking. Molecular interaction analysis indicated that three amino acid residues (R120, R121 and K266) might play key roles in the catalysis of the two aphid FPPSs by forming hydrogen bonds with the diphosphate moiety of the allylic substrate. These in silico results were subsequently confirmed by site-directed mutagenesis and in vitro activity assay of the mutant enzymes, in which each of the single mutations R120G, R121G and K266I abolished the activities of the two FPPSs. This study contributes to our understanding of the catalytic mechanism of farnesyl diphosphate synthases.