Functional analysis and molecular docking identify two active short-chain prenyltransferases in the green peach aphid, Myzus persicae.
Short-chain prenyltransferases are responsible for biosynthesis of the C(10)-C(20) precursors of a variety of isoprenoids. We previously isolated two different short-chain prenyltransferases from the green peach aphid, Myzus persicae (MpIPPS1 and MpIPPS2). In this study, the activity of the two aphid prenyltransferases was analyzed in vitro. Kinetic analysis using recombinant enzymes showed that both prenyltransferases could efficiently catalyze the formation of C(10) geranyl diphosphate (GPP) and C(15) farnesyl diphosphate (FPP) from the C(5) substrates isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), and MpIPPS2 had higher catalytic activity than MpIPPS1. Product analysis by gas chromatography-mass spectrometry demonstrated that FPP was generated as the major product, but GPP could be detected at low enzyme concentrations. Molecular docking revealed that MpIPPS2 had higher binding affinity with the substrates DMAPP, IPP, and GPP than MpIPPS1, which supported the experimentally determined kinetic parameters. Molecular docking also identified an amino acid residue (K266) critical to the catalytic activity of both MpIPPS1 and MpIPPS2. This prediction was subsequently confirmed by site-directed mutagenesis, in which a point mutation (K266I) abolished the activity of both MpIPPS1 and MpIPPS2. Our data illustrate that both aphid short-chain prenyltransferases are active forms, which is in contrast to the previously reported results.
Department of Entomology, China Agricultural University, Beijing, China.
Yong-Lei
YL
Zhang
Zheng-Xi
ZX
Li
D000818:Animals; D001042:Aphids / Q000201:enzymology*; D004122:Dimethylallyltranstransferase / Q000737:chemistry / Q000378:metabolism*; D011756:Diphosphates / Q000737:chemistry / Q000378:metabolism; D004224:Diterpenes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D062105:Molecular Docking Simulation; D011106:Polyisoprenyl Phosphates / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D012717:Sesquiterpenes / Q000737:chemistry / Q000378:metabolism
D011756:Diphosphates; D004224:Diterpenes; D011106:Polyisoprenyl Phosphates; D011994:Recombinant Proteins; D012717:Sesquiterpenes; C511282:geranyl diphosphate; C004808:farnesyl pyrophosphate; D004122:Dimethylallyltranstransferase