Isoprenyl Diphosphate Synthases Research Articles

Iridoid biosynthesis in Chrysomelina larvae: Fat body produces early terpenoid precursors

Larvae of the Chrysomelina species Phaedon cochleariae and Gastrophysa viridula produce monoterpenoids (iridoids) to defend themselves against predatory attacks by presenting the toxins upon attack as droplets on the top of nine pairs of dorsal glands. Although the conversion of 8-hydroxygeraniol-8- O- β- d-glucoside into the iridoids in the glandular reservoir has been studied in detail, the synthesis of the glucosidically bound precursor received only limited attention. We compared larvae of the two iridoid producing species with those of Chrysomela populi, a sequestering species producing salicylaldehyde, in terms of the key enzymes 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) and isoprenyl diphosphate synthases involved in the biosynthesis of the iridoid precursor. Increased HMGR transcript abundance, high HMGR activity and accumulation of geraniol indicating geranyl diphosphate synthase activity was observed only in the fat body of the iridoid producing larvae in comparison to other larval tissues and to the tested tissues of C. populi. These results correlate with the identification of glucosidically bound 8-hydroxygeraniol in the fat body of the iridoid producers. We suggest that in P. cochleariae and G. viridula glucosidically bound 8-hydroxygeraniol is produced by the fat body and transferred via the hemolymph into the glandular reservoir for further conversion into iridoids.

Investigation of the Catalytic Mechanism of Farnesyl Pyrophosphate Synthase by Computer Simulation

Farnesyl pyrophosphate synthase (FPPS) catalyses the formation of a key cellular intermediate in isoprenoid metabolic pathways, farnesyl pyrophosphate, by the sequential head-to-tail condensation of two molecules of isopentenyl diphosphate (IPP) with dimethylallyl diphosphate (DMAPP). Recently, FPPS has been shown to represent an important target for the treatment of parasitic diseases such as Chagas disease and African trypanosomiasis. Bisphosphonates, pyrophosphate analogues in which the oxygen bridge between the two phosphorus atoms has been replaced by a carbon substituted with different side chains, are able to inhibit the FPPS enzyme. Moreover, nitrogen-containing bisphosphonates have been proposed as carbocation transition state analogues of FPPS. On the basis of structural and kinetic data, different catalytic mechanisms have been proposed for FPPS. By analyzing different reaction coordinates we propose that the reaction occurs in one step through a carbocationic transition state and the subsequent transfer of a hydrogen atom from IPP to the pyrophosphate moiety of DMAPP. Moreover, we have analyzed the role of the active site amino acids on the activation barrier and the reaction mechanism. The structure of the active site is well conserved in the isoprenyl diphosphate synthase family; thus, our results are relevant for the understanding of this important class of enzymes and for the design of more potent and specific inhibitors for the treatment of parasitic diseases.

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

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.

Purification, Enzymatic Characterization, and Inhibition of theZ-Farnesyl Diphosphate Synthase from Mycobacterium tuberculosis

We have recently shown that open reading frame Rv1086 of the Mycobacterium tuberculosis H37Rv genome sequence encodes a unique isoprenyl diphosphate synthase. The product of this enzyme, omega,E,Z-farnesyl diphosphate, is an intermediate for the synthesis of decaprenyl phosphate, which has a central role in the biosynthesis of most features of the mycobacterial cell wall, including peptidoglycan, arabinan, linker unit galactan, and lipoarabinomannan. We have now purified Z-farnesyl diphosphate synthase to near homogeneity using a novel mycobacterial expression system. Z-Farnesyl diphosphate synthase catalyzed the addition of isopentenyl diphosphate to omega,E-geranyl diphosphate or omega,Z-neryl diphosphate yielding omega,E,Z-farnesyl diphosphate and omega,Z,Z-farnesyl diphosphate, respectively. The enzyme has an absolute requirement for a divalent cation, an optimal pH range of 7-8, and K(m) values of 124 micrometer for isopentenyl diphosphate, 38 micrometer for geranyl diphosphate, and 16 micrometer for neryl diphosphate. Inhibitors of the Z-farnesyl diphosphate synthase were designed and chemically synthesized as stable analogs of omega,E-geranyl diphosphate in which the labile diphosphate moiety was replaced with stable moieties. Studies with these compounds revealed that the active site of Z-farnesyl diphosphate synthase differs substantially from E-farnesyl diphosphate synthase from pig brain (Sus scrofa).

Identification of a Short (C15) ChainZ-Isoprenyl Diphosphate Synthase and a Homologous Long (C50) Chain Isoprenyl Diphosphate Synthase in Mycobacterium tuberculosis

We report the cloning, overexpression, and partial characterization of two unique Z-isoprenyl diphosphate synthase homologs from Mycobacterium tuberculosis. The first enzyme, Rv1086, adds one isoprene unit to omega,E-geranyl diphosphate. The product, omega,E, Z-farnesyl diphosphate, is the putative substrate of the second enzyme, Rv2361c. This enzyme adds seven more isoprene units to omega, E,Z-farnesyl diphosphate and releases decaprenyl diphosphate. Both open reading frames were cloned from the M. tuberculosis H37Rv genome and overexpressed in M. smegmatis. Membrane and cytosol fractions from wild type and the two recombinant strains were assayed for [(14)C]isopentenyl diphosphate incorporation into isoprenyl diphosphates in the presence of various allylic isoprenyl diphosphate acceptors. Membrane fractions of recombinant cells overexpressing Rv2361c incubated with farnesyl diphosphate showed a 10-fold increase of [(14)C]isopentenyl diphosphate incorporation into decaprenyl diphosphate. Membrane fractions of recombinant cells overexpressing Rv1086 incubated with geranyl diphosphate showed a 5-fold increase of [(14)C]isopentenyl diphosphate incorporation into farnesyl diphosphate. Analysis of the stereochemistry revealed that all of the overexpressed farnesyl diphosphate was in the omega,E, Z-configuration. This is the first description of a short chain isoprenyl diphosphate synthase that generates products with Z-stereochemistry. Previously, all known short chain isoprenyl diphosphate synthases catalyze the synthesis of products with E-stereochemistry.

Chain elongation in the isoprenoid biosynthetic pathway

Isoprenyl diphosphate synthases catalyze addition of allylic diphosphate primers to the isoprene unit in isopentenyl diphosphate to produce polyisoprenoid diphosphates with well defined chain lengths. Phylogenetic correlations suggest that the synthases which catalyze formation of isoprenoid diphosphates with ( E) double bonds have evolved from a common ancestor. X-ray crystallographic studies of farnesyl diphosphate synthase in conjunction with site-directed mutagenesis have provided important new information about the residues involved in binding and catalysis and the source of chain length selectivity for the enzymes that catalyze chain elongation.

Isolation and Characterization of idsA: The Gene for the Short Chain Isoprenyl Diphosphate Synthase from Methanobacterium thermoautotrophicum

The gene that encodes the bifunctional short chain isoprenyl diphosphate synthase (idsA) for synthesis of farnesyl diphosphate and geranylgeranyl diphosphate in Methanobacterium thermoautotrophicum, a strict archaebacterial anaerobe, was isolated from a genomic DNA library by colony-lift hybridization and sequenced. Amino acid sequences were obtained for the N-terminus of the enzyme and for internal peptide fragments generated by proteolysis and treatment with cyanogen bromide. Degenerate primers based on the amino acid sequences were used in PCR to synthesize a 220-bp probe from genomic DNA. The probe was radiolabeled and used to isolate idsA. DNA sequencing revealed a 975-bp open reading frame located within an operon. The encoded 325-amino-acid protein contained five conserved regions found in eubacterial and eukaryotic farnesyl diphosphate and geranylgeranyl diphosphate synthases, including aspartate-rich motifs commonly found in prenyltransferases.

Crystal structure of recombinant farnesyl diphosphate synthase at 2.6-A resolution.

The synthesis of farnesyl diphosphate (FPP), a key intermediate in the isoprenoid biosynthetic pathway required for the synthesis of cholesterol and in the formation of prenylated proteins, is catalyzed by the enzyme farnesyl diphosphate synthase (FPS). The crystal structure of avian recombinant FPS, the first three-dimensional structure for any prenyltransferase, was determined to 2.6-A resolution. The enzyme exhibits a novel fold composed entirely of alpha-helices joined by connecting loops. The enzyme's most prominent structural feature is the arrangement of 10 core helices around a large central cavity. Two aspartate-rich sequences that are highly conserved among the isoprenyl diphosphate synthase family of prenyltransferases, and are essential for enzymatic activity, were found on opposite walls of this cavity, with the aspartate side chains approximately 12 A apart and facing each other. The location and metal ion binding properties of these sequences suggest that the conserved aspartate residues participate in substrate binding of catalysis.

Isoprenyl diphosphate synthases: Protein sequence comparisons, a phylogenetic tree, and predictions of secondary structure

Isoprenyl diphosphate synthases are ubiquitous enzymes that catalyze the basic chain-elongation reaction in the isoprene biosynthetic pathway. Pairwise sequence comparisons were made for 6 farnesyl diphosphate synthases, 6 geranylgeranyl diphosphate synthases, and a hexaprenyl diphosphate synthase. Five regions with highly conserved residues, two of which contain aspartate-rich DDXX(XX)D motifs found in many prenyltransferases, were identified. A consensus secondary structure for the group, consisting mostly of alpha-helices, was predicted for the multiply aligned sequences from amino acid compositions, computer assignments of local structure, and hydropathy indices. Progressive sequence alignments suggest that the 13 isoprenyl diphosphate synthases evolved from a common ancestor into 3 distinct clusters. The most distant separation is between yeast hexaprenyl diphosphate synthetase and the other enzymes. Except for the chromoplastic geranylgeranyl diphosphate synthase from Capsicum annuum, the remaining farnesyl and geranylgeranyl diphosphate synthases segregate into prokaryotic/archaebacterial and eukaryotic families.