In vitro and in vivo characterization of a novel insect decaprenyl diphosphate synthase: a two-major step catalytic mechanism is proposed.
Medium- and long-chain polyprenyl diphosphate synthases (PDDSs) catalyze the synthesis of the side-chain prenyl tails of ubiquinones, which play critical physiological roles in all organisms. This class of enzymes has been extensively studied in bacteria, yeast, plants and mammals, but very little information about such enzymes is available in insects. Here we cloned the cDNAs encoding the two subunits of an aphid long-chain PDDS (designated as AgDPPS1 and AgDPPS2). AgDPPS1 and AgDPPS2 had an open reading frame of 1230 bp and 1275 bp, with a calculated isoelectric point of 8.13 and 6.28, respectively. Sequence alignment and phylogenetic analysis showed that the enzyme was a candidate decaprenyl diphosphate (DPP) synthase with two heterologous subunits. Recombinant expression and in vitro enzymatic assay revealed that the two subunits were essential for the activity of the enzyme that catalyzed the formation of a major intermediate product geranylgeranyl diphosphate. In vivo analysis of ubiquinone (UQ) by expressing the insect enzyme in Escherichia coli identified UQ-10. Our data suggested that the insect enzyme is a novel DPP synthase with a two-major step catalytic mechanism, which catalyzes the formation of DPP as the final product, with geranylgeranyl diphosphate as the major intermediate product. This is the first characterization of an insect long-chain DPPS that synthesizes the side-chain of coenzyme Q-10.
Department of Entomology, China Agricultural University, 2 Yuanmingyuan West Road, Beijing 100193, China.
Hao
H
Zhang
Zheng-Xi
ZX
Li
D019883:Alkyl and Aryl Transferases / Q000737:chemistry* / Q000145:classification / Q000235:genetics; D000818:Animals; D001042:Aphids / Q000201:enzymology* / Q000235:genetics; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D008401:Gas Chromatography-Mass Spectrometry; D019476:Insect Proteins / Q000737:chemistry* / Q000145:classification / Q000235:genetics; D010802:Phylogeny; D021122:Protein Subunits / Q000737:chemistry / Q000145:classification / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014451:Ubiquinone / Q000032:analysis
D019476:Insect Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D014451:Ubiquinone; D019883:Alkyl and Aryl Transferases; C039821:decaprenyl pyrophosphate synthetase
CoQ; Coenzyme Q; DL-1,4-dithiothreitol; DMAPP; DPP; DPPS; DTT; Decaprenyl diphosphate synthase; FARM; FPP; FPPS; GGPP; GGPPS; GPP; GST; HepPP; HepPPS; HexPP; HexPPS; IPP; IPTG; In vitro and in vivo enzymatic assay; OPP; OPPS; PDDS; Polyprenyl diphosphate synthase; SARM; SPP; SPPS; UQ; Ubiquinone; coenzyme Q; decaprenyl diphosphate; decaprenyl diphosphate synthase; dimethylallyl diphosphate; farnesyl diphosphate; farnesyl diphosphate synthase; geranyl diphosphate; geranylgeranyl diphosphate; geranylgeranyl diphosphate synthase; glutathione S-transferase; heptaprenyl diphosphate; heptaprenyl diphosphate synthase; hexaprenyl diphosphate; hexaprenyl diphosphate synthase; isopentenyl diphosphate; isopropyl thio-β-d-galactoside; octaprenyl diphosphate; octaprenyl diphosphate synthase; polyprenyl diphosphate synthase; solanesyl diphosphate; solanesyl diphosphate synthase; the first aspartate-rich motif; the second aspartate -rich motif; ubiquinone