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Publication Title | Heterodimeric geranyl(geranyl)diphosphate synthase from hop (Humulus lupulus) and the evolution of monoterpene biosynthesis

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Heterodimeric geranyl(geranyl)diphosphate synthase from hop (Humulus lupulus) and the evolution of monoterpene biosynthesis

Guodong Wang and Richard A. Dixon1

Plant Biology Division, Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK 73401 Contributed by Richard A. Dixon, April 14, 2009 (sent for review December 5, 2008)

Myrcene, which accounts for 30–50% of the essential oil in hop (Humulus lupulus L.) trichomes, derives from geranyl diphosphate (GPP), the common precursor of monoterpenes. Full-length se- quences of heterodimeric GPP synthase small subunit (GPPS.SSU, belonging to the SSU I subfamily) and large subunit (LSU) cDNAs were mined from a hop trichome cDNA library. The SSU was inactive, whereas the LSU produced GPP, farnesyl diphosphate, and geranylgeranyl diphosphate (GGPP) from dimethylallyl diphosphate and isopentenyl diphosphate in vitro. Coexpression of both subunits in Escherichia coli yielded a heterodimeric enzyme exhibiting altered ratios of GPP and GGPP synthase activities and greatly enhanced catalytic efficiency. Transcript analysis suggested that the heterodimeric geranyl(geranyl)diphosphate synthase [G(G)PPS] is involved in myrcene biosynthesis in hop trichomes. The critical role of the conserved CxxxC motif (where ‘‘x’’ can be any hydrophobic amino acid residue) in physical interactions between the 2 subunits was demonstrated by using site-directed mutagen- esis, and this motif was used in informatic searches to reveal a previously undescribed SSU subfamily (SSU II) present in both angiosperms and gymnosperms. The evolution and physiological roles of SSUs are discussed.

terpene biosynthesis trichome enzyme evolution subunit interactions

Monoterpenes represent a large family of plant natural products. They can be released from vegetative tissues to serve directly as toxic agents against herbivores or pathogens, or they can indirectly protect plants by attracting predators of attacking herbivores (1–3). Monoterpenes are also emitted from floral tissues to attract pollinators (4).

Geranyl diphosphate (GPP), the common precursor for monoterpenes, is formed by head-to-tail condensation of 1 molecule of isopentenyl diphosphate (IPP) and 1 molecule of dimethylallyl diphosphate (DMAPP) via the action of GPP synthase (GPPS; EC in plastids of plant cells. Het- erodimeric GPPSs have been described from angiosperms (5, 6), and homodimeric GPPSs have been functionally characterized from both angiosperms (7, 8) and gymnosperms (9, 10). Both subunits of homodimeric GPPSs and the large subunit of het- erodimeric GPPSs (GPPS.LSU) contain 2 aspartate-rich motifs, DD(X)2– 4D (where ‘‘X’’ is any amino acid), which are important in prenyl–substrate binding (11). However, the small subunit of heterodimeric GPPS (GPPS.SSU) lacks this motif and is cata- lytically inactive alone. The LSU of Mentha piperita GPPS is also inactive alone, whereas that from Antirrhinum majus can convert DMAPP and IPP to geranylgeranyl diphosphate (GGPP) in vitro (5, 6). In both cases, the inactive SSU functions as a ‘‘modifier’’ to change the chain length of the product of the LSU from GGPP(C20) to GPP(C10). However, the effect of SSU on the catalytic efficiency of an active LSU, and the mechanism of physical interaction between the 2 subunits of heterodimeric GPPSs have yet to be investigated.

Mentha SSU can bind to the phylogenetically distant gera- nylgeranyl diphosphate synthases (GGPPSs) from the gymno- sperms Taxus canadensis and Abies grandis to produce GPP as

the main product (12), and it has been suggested that both heterodimeric GPPS subunits might have evolved from a GGPPS based on the fact that diterpene biosynthesis evolution- arily predates monoterpene biosynthesis (13). However, failure to clone SSU I subfamily homologs from A. grandis or Picea abies via homology-based strategies (8, 9) led to the assumption that heterodimeric GPPS might exist only in angiosperm species.

Plant monoterpenes are derived from the plastid-localized methylerythritol phosphate pathway (14, 15), and monoterpene synthases of plant origin have an N-terminal plastid-localization signal peptide (16). On the basis of colocalization, it has been assumed that the homodimeric GPPS is responsible for the formation of GPP as the precursor of monoterpenes in Arabi- dopsis, whereas this function has been ascribed to the het- erodimeric GPPS in M. piperita and A. majus. Tissue-specific, developmental, and rhythmic changes in the mRNA and protein levels of GPPS.SSU, but not LSU, correlate with monoterpene emission (6), suggesting that SSU might play a key role in the regulation of monoterpene biosynthesis in A. majus flowers.

Cultivated hop (Humulus lupulus) is a dioecious perennial vine, and female flowers (commonly called cones) are used to add flavor during the brewing of beer. The monoterpene myr- cene accounts for 30–50% of the essential oil, depending on variety (17). Myrcene is synthesized and stored exclusively in lupulin glands (trichomes on the inner surface of the bracts of the female flowers). The tissue-specific and developmental expres- sion profiles of a hop monoterpene synthase suggested a primary role in myrcene formation (18).

We report here the functional expression of heterodimeric GPPS large and small subunits from hop. The hop SSU possesses no prenyltransferase activity alone, but it alters both the kinetics and product specificity of the LSU. Expression of the SSU, but not the LSU, is directly correlated with myrcene production. The CxxxC motif (where ‘‘x’’ can be a hydrophobic amino acid, such as alanine, leucine, isoleucine, valine, glycine, or serine) found in both subunits of the hop heterodimeric geranyl(geranyl)diphos- phate synthase [G(G)PPS] is crucial for physical interactions between the 2 subunits. Finally, biochemical and phylogenetic analyses revealed the existence of a previously undescribed class of heterodimeric G(G)PPS.SSUs with broad distribution in both angiosperms and gymnosperms.

Results and Discussion

Isolation and Analysis of Hop GPPS.SSU and GPPS.LSU cDNA Clones.

Searching the previously described hop trichome EST database (18) consisting of 3,619 unigenes led to the identification of a

Author contributions: G.W. and R.A.D. designed research; G.W. performed research; G.W. contributed new reagents/analytic tools; R.A.D. analyzed data; and R.A.D. wrote the paper.

The authors declare no conflict of interest.

Data deposition: The sequences reported in this paper have been deposited in the GenBank database {accession nos. FJ455406 [hop G(G)PPS small subunit] and FJ455407 [hop G(G)PPS large subunit]}.

1To whom correspondence should be addressed. E-mail:

This article contains supporting information online at 0904069106/DCSupplemental.

9914–9919 PNAS June 16, 2009 vol. 106 no. 24 cgi doi 10.1073 pnas.0904069106

Image | Heterodimeric geranyl(geranyl)diphosphate synthase from hop (Humulus lupulus) and the evolution of monoterpene biosynthesis

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