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

Abstract

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 sequences 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 mutagenesis, 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

Chrysanthemyl Diphosphate Synthase Operates in Planta as a Bifunctional Enzyme with Chrysanthemol Synthase Activity

“…In tobacco plants overexpressing three monoterpene synthases (CaMV35S promoter), the total level of additional monoterpenes emitted from leaves reached up to 30 ng h Ϫ1 g Ϫ1 FW (37), and plants overexpressing patchoulol synthase emitted patchoulol at levels of 50 -100 ng h Ϫ1 g Ϫ1 FW (6). The high efficiency of chrysanthemol production in plants suggests the possible presence of an endogenous phosphatase that efficiently hydrolyzes CPP or the presence of an endogenous cofactor that potentiates CDS activity (38,39). Our experiments and existing literature seem to best support the latter possibility for the reasons given below.…”

“…13), can be estimated to be around 30 M, assuming a regression coefficient of 1 g ml Ϫ1 for leaf fresh weight to leaf volume (44), a chloroplast volume of 20% of total cell volume in mature leaf cells (45), and 60% of DMAPP to be found in the chloroplast (46). Third, inactive GDSs have been shown before to be activated by the formation of a heterodimer with geranylgeranyldiphospate synthase (39,47,48). The small subunits are usually inactive in the in vitro assays, and the big subunits are either inactive (35,49) or function as well as homodimeric geranylgeranyl diphosphate synthases (39,47,48).…”

“…Third, inactive GDSs have been shown before to be activated by the formation of a heterodimer with geranylgeranyldiphospate synthase (39,47,48). The small subunits are usually inactive in the in vitro assays, and the big subunits are either inactive (35,49) or function as well as homodimeric geranylgeranyl diphosphate synthases (39,47,48). Two of four geranylgeranyl diphosphate synthase-like enzymes of tobacco could interact in tobacco with the small subunit from snapdragon (Antirrhinum majus) (48).…”

A recruiting protein of geranylgeranyl diphosphate synthase controls metabolic flux toward chlorophyll biosynthesis in rice

“…of this subfamily are catalytically inactive but are able to bind GGPPS and modulate its product specificity from GGPP to GPP (11). Recombinant Os02g44780 protein (SI Appendix, Fig.…”

“…Although the SSU II subfamily is present in both angiosperms and gymnosperms, to date, only one member of this subfamily was functionally characterized (11). It has been shown that Arabidopsis SSU II, although forming heterodimers with GGPPS, modifies the product profile and promotes the GPPS activity of GGPPS (11).…”