Purification, properties and heteromeric association of type-1 and type-2 lepidopteran farnesyl diphosphate synthases.
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.
Sen
Stephanie E
SE
Department of Chemistry, Indiana U.-Purdue U. Indianapolis (IUPUI), 402 North Blackford Street, Indianapolis, IN 46202, USA. sen@chem.iupui.edu
Cusson
Michel
M
Trobaugh
Corey
C
Béliveau
Catherine
C
Richard
Thenesha
T
Graham
Wendy
W
Mimms
Adrian
A
Roberts
Gregory
G
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
D019476:Insect Proteins; D011993:Recombinant Fusion Proteins; D051228:Geranyltranstransferase