Interaction with the Small Subunit of Geranyl Diphosphate Synthase Modifies the Chain Length Specificity of Geranylgeranyl Diphosphate Synthase to Produce Geranyl Diphosphate

Abstract

Geranyl diphosphate synthase belongs to a subgroup of prenyltransferases, including farnesyl diphosphate synthase and geranylgeranyl diphosphate synthase, that catalyzes the specific formation, from C 5 units, of the respective C 10 , C 15 , and C 20 precursors of monoterpenes, sesquiterpenes, and diterpenes. Unlike farnesyl diphosphate synthase and geranylgeranyl diphosphate synthase, which are homodimers, geranyl diphosphate synthase from Mentha is a heterotetramer in which the large subunit shares functional motifs and a high level of amino acid sequence identity (56 -75%) with geranylgeranyl diphosphate synthases of plant origin. The small subunit, however, shares little sequence identity with other isoprenyl diphosphate synthases; yet it is absolutely required for geranyl diphosphate synthase catalysis. Coexpression in Escherichia coli of the Mentha geranyl diphosphate synthase small subunit with the phylogenetically distant geranylgeranyl diphosphate synthases from Taxus canadensis and Abies grandis yielded a functional hybrid heterodimer that generated geranyl diphosphate as product in each case. These results indicate that the geranyl diphosphate synthase small subunit is capable of modifying the chain length specificity of geranylgeranyl diphosphate synthase (but not, apparently, farnesyl diphosphate synthase) to favor the production of C 10 chains. Comparison of the kinetic behavior of the parent prenyltransferases with that of the hybrid enzyme revealed that the hybrid possesses characteristics of both geranyl diphosphate synthase and geranylgeranyl diphosphate synthase.A subgroup of isoprenyl diphosphate synthases, referred to as the "short-chain prenyltransferases," consists of geranyl diphosphate (GPP 1 ; C 10 ) synthase, farnesyl diphosphate (FPP; C 15 ) synthase, and geranylgeranyl diphosphate (GGPP; C 20 ) synthase. These enzymes provide the acyclic branch point intermediates for the biosynthesis of numerous terpenoids, including monoterpenes, sesquiterpenes, diterpenes, triterpenes, tetraterpenes, and polyterpenes such as natural rubber. GGPP synthase and FPP synthase occur nearly ubiquitously in plants, animals, and bacteria (1). GPP synthase appears to be of much more limited distribution in nature, having been identified most frequently in essential oil (monoterpene)-producing plants (2). Most isoprenyl diphosphate synthases, including the short-chain prenyltransferases, catalyze the divalent metal iondependent 1Ј-4 condensation of isopentenyl diphosphate (IPP) with an allylic prenyl diphosphate cosubstrate (3), and they are distinguished by the specific chain length and double bond geometry at C2-C3 of the prenyl diphosphate product generated (Fig. 1). Thus, GPP synthase catalyzes a single condensation of IPP with dimethylallyl diphosphate (DMAPP) to form, specifically, GPP (C 10 ). FPP synthase and GGPP synthase catalyze sequential condensations of IPP with an allylic primer (i.e. DMAPP, GPP, or FPP, as appropriate) to form the respective C 15 and C 20 elongation products. Reaction paramet...

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

“…The hop SSU and LSU genes were N-terminally truncated (Fig. S1 A) to aid solubility (9,12) and were subcloned into appropriate vectors (see Materials and Methods, SI Materials and Methods, and Table S1), followed by expression in appropriate Escherichia coli strains for analysis of the biochemical properties of the recombinant proteins.…”

“…Mentha SSU can bind to the phylogenetically distant geranylgeranyl diphosphate synthases (GGPPSs) from the gymnosperms 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 evolutionarily 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.…”

The Small Subunit of Snapdragon Geranyl Diphosphate Synthase Modifies the Chain Length Specificity of Tobacco Geranylgeranyl Diphosphate Synthase in Planta

“…The first heterodimeric GPPS containing large and small subunits was isolated from peppermint (Mentha piperita) glandular trichomes via enzyme purification and protein sequencing (Burke et al, 1999). While both proteins belong to the trans-prenyltransfrease family, the large subunit was similar to bona fide geranylgeranyl diphopsphate synthases (GGPPSs) (Burke et al, 1999;Burke and Croteau, 2002a), and the small subunit exhibited only a low level of homology to existing prenyltransferases (Burke et al, 1999). Both subunits were catalytically inactive when expressed alone in Escherichia coli but formed an active GPPS when coexpressed together (Burke et al, 1999).…”

A Modular Approach for Facile Biosynthesis of Labdane-Related Diterpenes

“…5 To minimize the number of plasmids required for these metabolic engineering efforts, rAgGGPS was introduced into the dual gene expression vector pACYCDuet, creating a base pGG plasmid (see Supporting Information). For initial proof-of-concept studies a previously reported pseudomature version of the bifunctional class II/I diterpene cyclase from grand fir, abietadiene synthase (rAgAS) 6 was added to pGG to create pGGAS.…”