title: Purification, properties and heteromeric association of type-1 and type-2 lepidopteran farnesyl diphosphate synthases.
abstract: Two forms of farnesyl diphosphate synthase (FPPS) from the spruce budworm, Choristoneura fumiferana, and one from the armyworm Pseudaletia unipuncta, have been cloned and their catalytic properties assessed. The type-2 FPPS of C. fumiferana (CfFPPS2) was efficient in the prenyl coupling of DMAPP or GPP with [(14)C]IPP, producing FPP as its final product; however, type-1 FPPSs (CfFPPS1, PuFPPS1, as well as Agrotis ipsilon FPPS1) were essentially inactive. A variety of purification methods was employed to purify the type-1 enzymes. Under mild chromatographic conditions, the isolated type-1 enzyme showed modest activity, but was apparently contaminated with endogenous prenyltransferase derived from the Escherichia coli host cells. Similarly, unpurified extracts of PuFPPS1 expressed in an E. coli FPPS-null mutant, had low FPPS activity. When equimolar amounts of homogenous CfFPPS1 and CfFPP2 were combined, a sharp synergistic enhancement of activity was observed, and the coupling of several homologous substrates, which are precursors to ethyl-branched JHs, was enhanced. Association between CfFPPS1 and CfFPPS2 was confirmed by both protein interaction chromatography and competitive ELISA. These data suggest that type-1 and type-2 FPPSs can form a heteromer, which may play a role in sesquiterpene biosynthesis, such as JH homologue formation, in moths.
authors.affiliation: Department of Chemistry, Indiana U.-Purdue U. Indianapolis (IUPUI), 402 North Blackford Street, Indianapolis, IN 46202, USA. sen@chem.iupui.edu
authors.forename: Stephanie E
authors.initials: SE
authors.lastname: Sen
authors.forename: Michel
authors.initials: M
authors.lastname: Cusson
authors.forename: Corey
authors.initials: C
authors.lastname: Trobaugh
authors.forename: Catherine
authors.initials: C
authors.lastname: Béliveau
authors.forename: Thenesha
authors.initials: T
authors.lastname: Richard
authors.forename: Wendy
authors.initials: W
authors.lastname: Graham
authors.forename: Adrian
authors.initials: A
authors.lastname: Mimms
authors.forename: Gregory
authors.initials: G
authors.lastname: Roberts
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D051228:Geranyltranstransferase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D019476:Insect Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007915:Lepidoptera / Q000201:enzymology*; D008969:Molecular Sequence Data; D025941:Protein Interaction Mapping; D011993:Recombinant Fusion Proteins; D016415:Sequence Alignment
chemical_list: D019476:Insect Proteins; D011993:Recombinant Fusion Proteins; D051228:Geranyltranstransferase