Isolation and functional expression of an animal geranyl diphosphate synthase and its role in bark beetle pheromone biosynthesis

Abstract

Geranyl diphosphate synthase (GPPS) catalyzes the condensation of dimethylallyl diphosphate and isopentenyl diphosphate to form geranyl diphosphate. Geranyl diphosphate is the precursor of monoterpenes, a large family of natural occurring C(10) compounds predominantly found in plants. Similar to plants but unique to animals, some bark beetle genera (Coleoptera: Scolytidae) produce monoterpenes that function in intraspecific chemical communication as aggregation and dispersion pheromones. The release of monoterpene aggregation pheromone mediates host colonization and mating. It has been debated whether these monoterpene pheromone components are derived de novo through the mevalonate pathway or result from simple modifications of dietary precursors. The data reported here provide conclusive evidence for de novo biosynthesis of monoterpene pheromone components from bark beetles. We describe GPPS in the midgut tissue of pheromone-producing male Ips pini. GPPS expression levels are regulated by juvenile hormone III, similar to other mevalonate pathway genes involved in pheromone biosynthesis. In addition, GPPS transcript is almost exclusively expressed in the anterior midgut of male I. pini, the site of aggregation pheromone biosynthesis. The recombinant enzyme was functionally expressed and produced geranyl diphosphate as its major product. The three-dimensional model structure of GPPS shows that the insect enzyme has the sequence structural motifs common to E-isoprenyl diphosphate synthases.

Fig. 1.

Abbreviated pathway of de novo biosynthesis defining the reaction catalyzed by GPPS in production of monoterpene pheromone components of male I. pini.

Fig. 2.

The cDNA coding region of GPPS is 416 amino acids. The conserved sequence domains (I–V) are underlined. The aspartate-rich motifs, common to all-E-isoprenyl diphosphate synthases, are bold and underlined. A peroxisomal targeting signaling sequence (italicized and underlined) is present at the N terminus.

Fig. 3.

Semiquantitative RT-PCR shows the tissue distribution of GPPS transcript from JH III- and acetone-treated male I. pini (head, H; anterior midgut, AM; posterior midgut, PM; hindgut, HG; fat body, FB; appendages, AP; carcass, C).

Fig. 4.

Radio-HPLC analyses of short-chain isoprenyl diphosphate products. Products are from midgut homogenates of JH III-treated male I. pini (A) and the recombinant-truncated GPPS (B).

Fig. 5.

Three-dimensional I. pini GPPS model structure and the avian FPPS crystal structure (1UBY). (A) GPPS model and avian crystal structures are superpositioned with DMAPP bound at the FARM region in the avian enzyme. The GPPS-extended N-terminal tail is not resolved in the avian crystal structure. (B) Catalytic Asp and Arg residues (avian FPPS, D₁₁₇,D₁₂₁,R₁₂₆; I. pini GPPS, D₁₅₇,D₁₆₁,R₁₆₆) are shown at the allylic binding site of the FARM region in the avian FPPS crystal structure and the I. pini GPPS model structure. The alpha helices are represented as barrels and the loop regions as tubes.

Fig. 6.

Neighbor-joining trees based on sequence similarity of short-chain E-IPPSs. Sequence comparisons of short-chain enzymes from plants and animals (A) and insects (B).