title: Cloning, expression and characterization of an insect geranylgeranyl diphosphate synthase from Choristoneura fumiferana.
abstract: Geranylgeranyl diphosphate synthase (GGPPS) catalyzes the condensation of the non-allylic diphosphate, isopentenyl diphosphate (IPP; C5), with allylic diphosphates to generate the C20 prenyl chain (GGPP) used for protein prenylation and diterpenoid biosynthesis. Here, we cloned the cDNA of a GGPPS from the spruce budworm, Choristoneura fumiferana, and characterized the corresponding recombinant protein (rCfGGPPS). As shown for other type-III GGPPSs, rCfGGPPS preferred farnesyl diphosphate (FPP; C15) over other allylic substrates for coupling with IPP. Unexpectedly, rCfGGPPS displayed inhibition by its FPP substrate at low IPP concentration, suggesting the existence of a mechanism that may regulate intracellular FPP pools. rCfGGPPS was also inhibited by its product, GGPP, in a competitive manner with respect to FPP, as reported for human and bovine brain GGPPSs. A homology model of CfGGPPS was prepared and compared to human and yeast GGPPSs. Consistent with its enzymological properties, CfGGPPS displayed a larger active site cavity that can accommodate the binding of FPP and GGPP in the region normally occupied by IPP and the allylic isoprenoid tail, and the binding of GGPP in an alternate orientation seen for GGPP binding to the human protein. To begin exploring the role of CfGGPPS in protein prenylation, its transcripts were quantified by qPCR in whole insects, along with those of other genes involved in this pathway. CfGGPPS was expressed throughout insect development and the abundance of its transcripts covaried with that of other prenylation-related genes. Our qPCR results suggest that geranylgeranylation is the predominant form of prenylation in whole C. fumiferana.
authors.affiliation: Département de biochimie, de microbiologie et de bio-informatique, Université Laval, Québec, QC G1V 0A6, Canada; Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, 1055 du P.E.P.S., C.P. 10380, Succ. Sainte-Foy, Québec, QC G1V 4C7, Canada.
authors.forename: Aline
authors.initials: A
authors.lastname: Barbar
authors.forename: Manon
authors.initials: M
authors.lastname: Couture
authors.forename: Stephanie E
authors.initials: SE
authors.lastname: Sen
authors.forename: Catherine
authors.initials: C
authors.lastname: Béliveau
authors.forename: Audrey
authors.initials: A
authors.lastname: Nisole
authors.forename: Marie
authors.initials: M
authors.lastname: Bipfubusa
authors.forename: Michel
authors.initials: M
authors.lastname: Cusson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D051231:Farnesyltranstransferase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D009036:Moths / Q000201:enzymology* / Q000254:growth & development; D017368:Protein Prenylation / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
chemical_list: D008024:Ligands; D011994:Recombinant Proteins; D051231:Farnesyltranstransferase
keywords: Geranylgeranyl diphosphate synthase; Geranylgeranylation; Homology modeling; Product inhibition; Protein prenylation; Substrate inhibition