In vitro and in vivo characterization of a novel insect decaprenyl diphosphate synthase: A two-major step catalytic mechanism is proposed

• Published in: Biochemical and biophysical research communications Vol. 442; no. 1-2; pp. 105 - 111
• Main Authors: Zhang, Hao, Li, Zheng-Xi
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 06.12.2013
• Subjects: Alkyl and Aryl Transferases - chemistry; Alkyl and Aryl Transferases - classification; Alkyl and Aryl Transferases - genetics; Animals; Aphids - enzymology; Aphids - genetics; Catalysis; Chromatography, High Pressure Liquid; Cloning, Molecular; Coenzyme Q; Decaprenyl diphosphate synthase; Gas Chromatography-Mass Spectrometry; In vitro and in vivo enzymatic assay; Insect Proteins - chemistry; Insect Proteins - classification; Insect Proteins - genetics; Phylogeny; Polyprenyl diphosphate synthase; Protein Subunits - chemistry; Protein Subunits - classification; Protein Subunits - genetics; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; Ubiquinone; Ubiquinone - analysis; GGPP; OPPS; CoQ; DPP; DTT; GPP; SPPS; FPP; IPP; Coenzyme Q; OPP; SPP; Ubiquinone; HepPP; FARM; In vitro and in vivo enzymatic assay; Polyprenyl diphosphate synthase; HexPPS; UQ; Decaprenyl diphosphate synthase; HexPP; HepPPS; PDDS; GST; FPPS; DPPS; SARM; IPTG; GGPPS; DMAPP; farnesyl diphosphate; DL-1,4-dithiothreitol; hexaprenyl diphosphate synthase; hexaprenyl diphosphate; decaprenyl diphosphate; isopropyl thio-β-d-galactoside; farnesyl diphosphate synthase; geranylgeranyl diphosphate synthase; heptaprenyl diphosphate synthase; geranylgeranyl diphosphate; octaprenyl diphosphate synthase; octaprenyl diphosphate; heptaprenyl diphosphate; dimethylallyl diphosphate; the first aspartate-rich motif; geranyl diphosphate; isopentenyl diphosphate; solanesyl diphosphate synthase; glutathione S-transferase; solanesyl diphosphate; the second aspartate -rich motif
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2013.11.025

•The two subunits of the novel enzyme are essential for the activity of the enzyme.•The enzyme catalyzes the formation of a major intermediate product.•The enzyme functions with a two-major step catalytic mechanism. 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 1230bp and 1275bp, 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.