Geranyl diphosphate synthase: cloning, expression, and characterization of this prenyltransferase as a heterodimer

Abstract

Geranyl diphosphate synthase, which catalyzes the condensation of dimethylallyl diphosphate and isopentenyl diphosphate to geranyl diphosphate, the key precursor of monoterpene biosynthesis, was purified from isolated oil glands of spearmint. Peptide fragments generated from the pure proteins of 28 and 37 kDa revealed amino acid sequences that matched two cDNA clones obtained by random screening of a peppermint-oil gland cDNA library. The deduced sequences of both proteins showed some similarity to existing prenyltransferases, and both contained a plastid-targeting sequence. Expression of each cDNA individually yielded no detectable prenyltransferase activity; however, coexpression of the two together produced functional geranyl diphosphate synthase. Antibodies raised against each protein were used to demonstrate that both subunits were required to produce catalytically active native and recombinant enzymes, thus confirming that geranyl diphosphate synthase is a heterodimer.

Figure 1

Mechanism of the prenyltransferase reaction illustrating the condensations catalyzed by GPP synthase, FPP synthase, and GGPP synthase.

Figure 2

Purification of GPP synthase from spearmint-oil gland secretory cells, product identification, and immunoblotting. (A) Anion-exchange chromatography of the prenyltransferase activity showing separation of FPP synthase (eluting at 90 mM KCl, region 1) from GPP synthase (eluting at 200 mM KCl, region 2). Absorbance at A₂₈₀, the KCl gradient, and the prenyltransferase assay values (in dpm) are indicated. (B) Radio-GC separation of the labeled dephosphorylated reaction products of FPP synthase (region 1) and GPP synthase (region 2). The upper tracing is the detector response to the authentic standards separated on the polydimethylsiloxane column: isopentenol (peak 1), dimethylallyl alcohol (peak 2), linalool (peak 3), nerol (peak 4), geraniol (peak 5), cis-nerolidol (peak 6), trans-nerolidol (peak 7), mixture of cis- and trans-farnesol (peak 8), and geranylgeraniol (peak 9). (C) Immunodetection of the large and small subunits of the native GPP synthase in the corresponding fractions separated by anion-exchange chromatography (A) using the antibodies raised against the recombinant proteins expressed from pMp13.18 and pMp23.10. The marker (Left) is carbonic anhydrase (29 kDa).

Figure 3

Alignment of the deduced amino acid sequences of pMp13.18 and pMp23.10 to each other and to their nearest homologues (A. thaliana GGPP synthase-like protein, AL035540, for pMp13.18, and Catharanthus roseus GGPP synthase, X92893, for pMp23.10). The DDXD motif of pMp13.18 is underlined in green. The red underlines indicate the DD(X)_2–4D motifs of the other sequences. Identical residues (three or more) are shown in black; similar residues are shaded.

Figure 4

Purification of recombinant GPP synthase expressed in E. coli, product identification and immunoblotting. (A) Anion-exchange chromatography of prenyltransferase activity (in dpm) expressed from pSBET13.18 (alone), pET23.10 (alone) and the combination of pSBET13.18-pET23.10, showing the separation of host-derived FPP synthase (eluting at 85 mM KCl, region 1) from GPP synthase (eluting at ≈200 mM KCl, region 2) obtained by coexpression. The KCl gradient is indicated by the dashed line. (B) Radio-GC separation of the labeled dephosphorylated reaction products of FPP synthase (1) and GPP synthase (2). The standards shown are the same as those described in Fig. 2B. (C) Immunodetection of the small and large subunits of GPP synthase (crude extracts) expressed alone and in combination (a), and of the functional heterodimer in the corresponding fractions (b) separated by anion-exchange chromatography (A), using the antibodies raised against the individual recombinant proteins expressed from pMp13.18 and pMp23.10. Empty vector controls (pSBET) and molecular mass markers, carbonic anhydrase (29 kDa) and ovalbumin (43 kDa), are included. Note that anti-pMp13.18 and anti-pMp23.10 detect other proteins (≈43 kDa) from the E. coli host (that are also detected by preimmune serum), and that, on coexpression of pMp13.18 and pMp23.10, truncated versions of both preproteins are produced.