Cloning, expression and characterization of a dipteran farnesyl diphosphate synthase.

Farnesyl diphosphate synthase (FPPS) of the dipteran Drosophila melanogaster has been cloned and its catalytic properties have been assessed. Analysis of the D. melanogaster genome and of ESTs indicates that FPPS is a single copy gene that produces two transcripts, which differ only by 5' extension. The cDNA of shorter and longer D. melanogaster FPPSs (DmFPPS-1a and DmFPPS-1b, respectively) were each subcloned into pET28a and expressed as an N-His6 fusion protein in BL21 E. coli cells. The DmFPPSs similarly catalyzed the coupling of the allylic substrates, GPP and DMAPP, with IPP, producing FPP as product. The longer protein was further characterized. The enzyme required divalent metal for activity, and was activated by 0.1% Triton X-100. Higher detergent concentration and the addition of glycerol, conditions that activate certain insect FPPSs, inhibited prenyl coupling by DmFPPS-1b. Although DmFPPS-1b does not efficiently couple homologous GPP compounds, homodimethylallyl diphosphate (HDMAPP), which is precursor to all homologous JH structures, was a reactive substrate.

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.

Juvenile hormone biosynthesis in M. sexta: substrate specificity of insect prenyltransferase utilizing homologous diphosphate analogs.

Analogs of dimethylallyl diphosphate (DMAPP) and geranyl diphosphate (GPP) were prepared and tested as potential substrates of prenyltransferase of the tobacco hornworm, Manduca sexta, and of a sesquiterpene synthase derived from pig liver. Enzyme derived from corpora allata homogenates of both the larval and adult stage of M. sexta coupled each of the DMAPP analogs to produce homologous geranyl and farnesyl diphosphate products in the order (Z)-3-ethyl>(Z)-3-n-propyl>(Z)-3-methyl (DMAPP)>(Z)-3-i-propyl(Z)-3-n-butyl. In competition studies, the ethyl and n-propyl analogs either enhanced or had no effect on DMAPP coupling, whereas the larger analogs were inhibitors. (Z)-7-ethyl and (2Z,6Z)-3,7-diethyl analogs of GPP were as good, if not better substrates of larval prenyltransferase, while the C-3 ethyl analog of GPP, which is precursor to an isomeric form of juvenile hormone (JH) that is not typically found in insects, was poorly coupled by the enzyme. While similarities were seen for whole-cell extracts derived from adult and larval M. sexta, adult prenyltransferase derived from cytosolic and 16,000xg pellet fractions displayed distinct competitive coupling of GPP and its homologs, suggesting differences in substrate specificity as a result of enzyme localization. In contrast to M. sexta, the pig liver enzyme poorly coupled each of the homologous DMAPP derivatives, and the homologous derivatives of GPP were less efficiently coupled than GPP. These results indicate that prenyltransferase in M. sexta possesses high steric latitude at the (Z)-C-3 and C-7 alkyl positions of DMAPP and GPP, respectively, in contrast to other animal prenyltransferases but in keeping with the enzyme's presumptive role in homologous JH metabolism.