Neryl Diphosphate Research Articles

Biosynthesis of nerol from glucose in the metabolic engineered Escherichia coli

In this study, nerol was biosynthesized in the metabolic engineered Escherichia coli from glucose for the first time. Firstly, the truncated neryl diphosphate synthase gene tNDPS1 was expressed that catalyzes isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) to form neryl diphosphate (NPP), and then the nerol synthase gene GmNES was co-expressed to synthesize the final product nerol from NPP. The engineered strain LZ001 accumulated 0.053 ± 0.015 mg/L of nerol. Secondly, the IDI1, MVD1, ERG8, ERG12, tHMG1 and ERG13 were co-expressed to increase the supply of IPP and DMAPP. Finally, the heterologous ERG10 gene was overexpressed, and the recombinant strain LZ005 produced 1.564 ± 0.102 mg/L of nerol in shaking-flask culture, which represents a 29.51-fold increase over LZ001 strain. This study shows the novel method for the biosynthesis of nerol and provides new metabolic engineering strategy for the production of terpenoids.

Mutational analysis and dynamic simulation of S-limonene synthase reveal the importance of Y573: Insight into the cyclization mechanism in monoterpene synthases

Monoterpene synthases carry out complex reactions to produce multiple products from a sole substrate, geranyl pyrophosphate (GPP). S-limonene synthase (LS) is a model monoterpene synthase that can be explored to understand the catalytic mechanism of these enzymes. In this study, we have identified an active site tyrosine residue (Y573) is crucial for the enzyme activity and mutational analysis indicates that both the aromatic ring and hydroxyl group are essential for the catalysis. Dynamic simulations found a hydrogen bond between Y573 and D496 and also a significant conformational change in the helical form of the LPP intermediate. Further mutagenesis suggested that this hydrogen bond is essential for catalysis. Sequence analysis suggested Y573 is completely conserved among cyclic monoterpene synthases but variable in acyclic enzymes, indicating this residue may be involved in cyclization. Subsequent studies by using neryl diphosphate (NPP) as the substrate ruled out the possibility that Y573 functions solely at the substrate isomerization step. Therefore, a more complicated role may be played by this residue. We proposed that Y573 may be involved in the earlier steps of the reaction, probably by controlling the conformation of the helical LPP intermediate. Our study provides important insights not only on the catalytic mechanism of LS, but also on the cyclization of monoterpene synthases in general.

Metabolic engineering of oleaginous yeast Yarrowia lipolytica for limonene overproduction.

BackgroundLimonene, a monocyclic monoterpene, is known for its using as an important precursor of many flavoring, pharmaceutical, and biodiesel products. Currently, d-limonene has been produced via fractionation from essential oils or as a byproduct of orange juice production, however, considering the increasing need for limonene and a certain amount of pesticides may exist in the limonene obtained from the citrus industry, some other methods should be explored to produce limonene.ResultsTo construct the limonene synthetic pathway in Yarrowia lipolytica, two genes encoding neryl diphosphate synthase 1 (NDPS1) and limonene synthase (LS) were codon-optimized and heterologously expressed in Y. lipolytica. Furthermore, to maximize limonene production, several genes involved in the MVA pathway were overexpressed, either in different copies of the same gene or in combination. Finally with the optimized pyruvic acid and dodecane concentration in flask culture, a maximum limonene titer and content of 23.56 mg/L and 1.36 mg/g DCW were achieved in the final engineered strain Po1f-LN-051, showing approximately 226-fold increase compared with the initial yield 0.006 mg/g DCW.ConclusionsThis is the first report on limonene biosynthesis in oleaginous yeast Y. lipolytica by heterologous expression of codon-optimized tLS and tNDPS1 genes. To our knowledge, the limonene production 23.56 mg/L, is the highest limonene production level reported in yeast. In short, we demonstrate that Y. lipolytica provides a compelling platform for the overproduction of limonene derivatives, and even other monoterpenes.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0626-7) contains supplementary material, which is available to authorized users.

Novel family of terpene synthases evolved from trans-isoprenyl diphosphate synthases in a flea beetle

Sesquiterpenes play important roles in insect communication, for example as pheromones. However, no sesquiterpene synthases, the enzymes involved in construction of the basic carbon skeleton, have been identified in insects to date. We investigated the biosynthesis of the sesquiterpene (6R,7S)-himachala-9,11-diene in the crucifer flea beetle Phyllotreta striolata, a compound previously identified as a male-produced aggregation pheromone in several Phyllotreta species. A (6R,7S)-himachala-9,11-diene-producing sesquiterpene synthase activity was detected in crude beetle protein extracts, but only when (Z,E)-farnesyl diphosphate [(Z,E)-FPP] was offered as a substrate. No sequences resembling sesquiterpene synthases from plants, fungi, or bacteria were found in the P. striolata transcriptome, but we identified nine divergent putative trans-isoprenyl diphosphate synthase (trans-IDS) transcripts. Four of these putative trans-IDSs exhibited terpene synthase (TPS) activity when heterologously expressed. Recombinant PsTPS1 converted (Z,E)-FPP to (6R,7S)-himachala-9,11-diene and other sesquiterpenes observed in beetle extracts. RNAi-mediated knockdown of PsTPS1 mRNA in P. striolata males led to reduced emission of aggregation pheromone, confirming a significant role of PsTPS1 in pheromone biosynthesis. Two expressed enzymes showed genuine IDS activity, with PsIDS1 synthesizing (E,E)-FPP, whereas PsIDS3 produced neryl diphosphate, (Z,Z)-FPP, and (Z,E)-FPP. In a phylogenetic analysis, the PsTPS enzymes and PsIDS3 were clearly separated from a clade of known coleopteran trans-IDS enzymes including PsIDS1 and PsIDS2. However, the exon-intron structures of IDS and TPS genes in P. striolata are conserved, suggesting that this TPS gene family evolved from trans-IDS ancestors.

Biosynthesis of the Diterpenoid Lycosantalonol via Nerylneryl Diphosphate in Solanum lycopersicum

We recently reported that three genes involved in the biosynthesis of monoterpenes in trichomes, a cis-prenyltransferase named neryl diphosphate synthase 1 (NDPS1) and two terpene synthases (TPS19 and TPS20), are present in close proximity to each other at the tip of chromosome 8 in the genome of the cultivated tomato (Solanum lycopersicum). This terpene gene “cluster” also contains a second cis-prenyltransferase gene (CPT2), three other TPS genes, including TPS21, and the cytochrome P450-oxidoreductase gene CYP71BN1. CPT2 encodes a neryneryl diphosphate synthase. Co-expression in E. coli of CPT2 and TPS21 led to the formation of the diterpene lycosantalene, and co-expression in E. coli of CPT2, TPS21 and CYP71BN1 led to the formation of lycosantalonol, an oxidation product of lycosantalene. Here we show that maximal expression of all three genes occurs in the petiolule part of the leaf, but little expression of these genes occurs in the trichomes present on the petiolules. While lycosantalene or lycosantalonol cannot be detected in the petiolules of wild-type plants (or anywhere else in the plant), lycosantalene and lycosantalonol are detected in petiolules of transgenic tomato plants expressing CPT2 under the control of the 35S CaMV promoter. These results suggest that lycosantalene and lycosantalonol are produced in the petiolules and perhaps in other tissues of wild-type plants, but that low rate of synthesis, controlled by the rate-limiting enzyme CPT2, results in product levels that are too low for detection under our current methodology. It is also possible that these compounds are further modified in the plant. The involvement of CPT2, TPS21 and CYP71BN1 in a diterpenoid biosynthetic pathway outside the trichomes, together with the involvement of other genes in the cluster in the synthesis of monoterpenes in trichomes, indicates that this cluster is further evolving into “sub-clusters” with unique biochemical, and likely physiological, roles.

Germacrene A synthase in yarrow (Achillea millefolium) is an enzyme with mixed substrate specificity: gene cloning, functional characterization and expression analysis.

Terpenoid synthases constitute a highly diverse gene family producing a wide range of cyclic and acyclic molecules consisting of isoprene (C5) residues. Often a single terpene synthase produces a spectrum of molecules of given chain length, but some terpene synthases can use multiple substrates, producing products of different chain length. Only a few such enzymes has been characterized, but the capacity for multiple-substrate use can be more widespread than previously thought. Here we focused on germacrene A synthase (GAS) that is a key cytosolic enzyme in the sesquiterpene lactone biosynthesis pathway in the important medicinal plant Achillea millefolium (AmGAS). The full length encoding gene was heterologously expressed in Escherichia coli BL21 (DE3), functionally characterized, and its in vivo expression was analyzed. The recombinant protein catalyzed formation of germacrene A with the C15 substrate farnesyl diphosphate (FDP), while acyclic monoterpenes were formed with the C10 substrate geranyl diphosphate (GDP) and cyclic monoterpenes with the C10 substrate neryl diphosphate (NDP). Although monoterpene synthesis has been assumed to be confined exclusively to plastids, AmGAS can potentially synthesize monoterpenes in cytosol when GDP or NDP become available. AmGAS enzyme had high homology with GAS sequences from other Asteraceae species, suggesting that multi-substrate use can be more widespread among germacrene A synthases than previously thought. Expression studies indicated that AmGAS was expressed in both autotrophic and heterotrophic plant compartments with the highest expression levels in leaves and flowers. To our knowledge, this is the first report on the cloning and characterization of germacrene A synthase coding gene in A. millefolium, and multi-substrate use of GAS enzymes.

Metabolic engineering of monoterpene biosynthesis in tomato fruits via introduction of the non-canonical substrate neryl diphosphate

Recently it was shown that monoterpenes in tomato trichomes (Solanum lycopersicum) are synthesized by phellandrene synthase 1 (PHS1) from the non-canonical substrate neryl diphosphate (NPP), the cis-isomer of geranyl diphosphate (GPP). As PHS1 accepts both NPP and GPP substrates forming different monoterpenes, it was overexpressed in tomato fruits to test if NPP is also available in a tissue highly active in carotenoid production. However, transgenic fruits overexpressing PHS1 produced only small amounts of GPP-derived PHS1 monoterpene products, indicating the absence of endogenous NPP. Therefore, NPP formation was achieved by diverting the metabolic flux from carotenoids via expression of tomato neryl diphosphate synthase 1 (NDPS1). NDPS1 transgenic fruits produced NPP-derived monoterpenes, including nerol, neral and geranial, while displaying reduced lycopene content. NDPS1 co-expression with PHS1 resulted in a monoterpene blend, including β-phellandrene, similar to that produced from NPP by PHS1 in vitro and in trichomes. Unexpectedly, PHS1×NDPS1 fruits showed recovery of lycopene levels compared to NDPS1 fruits, suggesting that redirection of metabolic flux is only partially responsible for the reduction in carotenoids. In vitro assays demonstrated that NPP serves as an inhibitor of geranylgeranyl diphosphate synthase, thus its consumption by PHS1 leads to recovery of lycopene levels. Monoterpenes produced in PHS1×NDPS1 fruits contributed to direct plant defense negatively affecting feeding behavior of the herbivore Helicoverpa zea and displaying antifungal activity against Botrytis cinerea. These results show that NPP-derived terpenoids can be produced in plant tissues; however, NPP has to be consumed to avoid negative impacts on plant metabolism.

Determination of Residues Responsible for Substrate and Product Specificity of Solanum habrochaites Short-Chain cis-Prenyltransferases

Isoprenoids are diverse compounds that have their biosynthetic origin in the initial condensation of isopentenyl diphosphate and dimethylallyl diphosphate to form C10 prenyl diphosphates that can be elongated by the addition of subsequent isopentenyl diphosphate units. These reactions are catalyzed by either cis-prenyltransferases (CPTs) or trans-prenyltransferases. The synthesis of volatile terpenes in plants typically proceeds through either geranyl diphosphate (C10) or trans-farnesyl diphosphate (C15), to yield monoterpenes and sesquiterpenes, respectively. However, terpene biosynthesis in glandular trichomes of tomato (Solanum lycopersicum) and related wild relatives also occurs via the cis-substrates neryl diphosphate (NPP) and 2Z,6Z-farnesyl diphosphate (Z,Z-FPP). NPP and Z,Z-FPP are synthesized by neryl diphosphate synthase1 (NDPS1) and Z,Z-farnesyl diphosphate synthase (zFPS), which are encoded by the orthologous CPT1 locus in tomato and Solanum habrochaites, respectively. In this study, comparative sequence analysis of NDPS1 and zFPS enzymes from S. habrochaites accessions that synthesize either monoterpenes or sesquiterpenes was performed to identify amino acid residues that correlate with the ability to synthesize NPP or Z,Z-FPP. Subsequent structural modeling, coupled with site-directed mutagenesis, highlighted the importance of four amino acids located within conserved domain II of CPT enzymes that form part of the second α-helix, for determining substrate and product specificity of these enzymes. In particular, the relative positioning of aromatic amino acid residues at positions 100 and 107 determines the ability of these enzymes to synthesize NPP or Z,Z-FPP. This study provides insight into the biochemical evolution of terpene biosynthesis in the glandular trichomes of Solanum species.

Identification and Characterization of a Novel Monoterpene Synthase from Soybean Restricted to Neryl Diphosphate Precursor

Terpenes are important defensive compounds against herbivores and pathogens. Here, we report the identification of a new monoterpene synthase gene, GmNES, from soybean. The transcription of GmNES was up-regulated in soybean plants that were infested with cotton leafworm (Prodenia litura), mechanically wounded or treated with salicylic acid (SA). Gas chromatography-mass spectrometry (GC-MS) analysis revealed that recombinant GmNES enzyme exclusively produced nerol, generated from a newly identified substrate for monoterpene synthase: neryl diphosphate (NPP). This finding indicates that GmNES is a nerol synthase gene in soybean. Subcellular localization using GFP fusions showed that GmNES localized to the chloroplasts. Transgenic tobacco overexpressing GmNES was generated. In dual-choice assays, the GmNES-expressing tobacco lines significantly repelled cotton leafworm. In feeding tests with transgenic plants, the growth and development of cotton leafworm were significantly retarded. This study confirms the ecological role of terpenoids and provides new insights into their metabolic engineering in transgenic plants.

Cloning of a sesquiterpene synthase from Lavandula x intermedia glandular trichomes

The essential oil (EO) of Lavandula is dominated by monoterpenes, but can also contain small amounts of sesquiterpenes, depending on species and environmental conditions. For example, the sesquiterpene 9-epi-caryophyllene can make up to 8 % of the EO in a few species, including those commercially propagated for EO production. Here, we report the cloning and functional characterization of 9-epi-caryophyllene synthase (LiCPS) from the glandular trichomes of Lavandula x intermedia, cv. Grosso. The 1,617 bp open reading frame of LiCPS, which did not encode a transit peptide, was expressed in Escherichia coli and the recombinant protein purified by Ni-NTA agarose affinity chromatography. The ca. 60 kDa recombinant protein specifically converted farnesyl diphosphate to 9-epi-caryophyllene. LiCPS also produced a few monoterpenes when assayed with the monoterpene precursor geranyl diphosphate (GPP), but--unlike most monoterpene synthases--was not able to derive detectable amounts of any products from the cis isomer of GPP, neryl diphosphate. The LiCPS transcripts accumulated in developing L. x intermedia flowers and were highly enriched in glandular trichomes, but were not detected in leaves suggesting that the transcriptional expression of this gene is spatially and developmentally regulated.

The tomato cis–prenyltransferase gene family

cis-prenyltransferases (CPTs) are predicted to be involved in the synthesis of long-chain polyisoprenoids, all with five or more isoprene (C5) units. Recently, we identified a short-chain CPT, neryl diphosphate synthase (NDPS1), in tomato (Solanum lycopersicum). Here, we searched the tomato genome and identified and characterized its entire CPT gene family, which comprises seven members (SlCPT1-7, with NDPS1 designated as SlCPT1). Six of the SlCPT genes encode proteins with N-terminal targeting sequences, which, when fused to GFP, mediated GFP transport to the plastids of Arabidopsis protoplasts. The SlCPT3-GFP fusion protein was localized to the cytosol. Enzymatic characterization of recombinant SlCPT proteins demonstrated that SlCPT6 produces Z,Z-FPP, and SlCPT2 catalyzes the formation of nerylneryl diphosphate while SlCPT4, SlCPT5 and SlCPT7 synthesize longer-chain products (C25-C55). Although no in vitro activity was demonstrated for SlCPT3, its expression in the Saccharomyces cerevisiae dolichol biosynthesis mutant (rer2) complemented the temperature-sensitive growth defect. Transcripts of SlCPT2, SlCPT4, SlCPT5 and SlCPT7 are present at low levels in multiple tissues, SlCPT6 is exclusively expressed in red fruit and roots, and SlCPT1, SlCPT3 and SlCPT7 are highly expressed in trichomes. RNAi-mediated suppression of NDPS1 led to a large decrease in β-phellandrene (which is produced from neryl diphosphate), with greater reductions achieved with the general 35S promoter compared to the trichome-specific MKS1 promoter. Phylogenetic analysis revealed CPT gene families in both eudicots and monocots, and showed that all the short-chain CPT genes from tomato (SlCPT1, SlCPT2 and SlCPT6) are closely linked to terpene synthase gene clusters.

Evolution of TPS20‐related terpene synthases influences chemical diversity in the glandular trichomes of the wild tomato relative Solanum habrochaites

A systematic screen of volatile terpene production in the glandular trichomes of 79 accessions of Solanum habrochaites was conducted and revealed the presence of 21 mono- and sesquiterpenes that exhibit a range of qualitative and quantitative variation. Hierarchical clustering identified distinct terpene phenotypic modules with shared patterns of terpene accumulation across accessions. Several terpene modules could be assigned to previously identified terpene synthase (TPS) activities that included members of the TPS-e/f subfamily that utilize the unusual cis-prenyl diphosphate substrates neryl diphosphate and 2z,6z-farnesyl diphosphate. DNA sequencing and in vitro enzyme activity analysis of TPS-e/f members from S. habrochaites identified three previously unassigned enzyme activities that utilize these cisoid substrates. These produce either the monoterpenes α-pinene and limonene, or the sesquiterpene 7-epizingiberene, with the in vitro analyses that recapitulated the trichome chemistry found in planta. Comparison of the distribution of S. habrochaites accessions with terpene content revealed a strong preference for the presence of particular TPS20 alleles at distinct geographic locations. This study reveals that the unusually high intra-specific variation of volatile terpene synthesis in glandular trichomes of S. habrochaites is due at least in part to evolution at the TPS20 locus.

A 1,6-Ring Closure Mechanism for (+)-δ-Cadinene Synthase?

Recombinant (+)-δ-cadinene synthase (DCS) from Gossypium arboreum catalyzes the metal-dependent cyclization of (E,E)-farnesyl diphosphate (FDP) to the cadinane sesquiterpene δ-cadinene, the parent hydrocarbon of cotton phytoalexins such as gossypol. In contrast to some other sesquiterpene cyclases, DCS carries out this transformation with >98% fidelity but, as a consequence, leaves no mechanistic traces of its mode of action. The formation of (+)-δ-cadinene has been shown to occur via the enzyme-bound intermediate (3R)-nerolidyl diphosphate (NDP), which in turn has been postulated to be converted to cis-germacradienyl cation after a 1,10-cyclization. A subsequent 1,3-hydride shift would then relocate the carbocation within the transient macrocycle to expedite a second cyclization that yields the cadinenyl cation with the correct cis stereochemistry found in (+)-δ-cadinene. An elegant 1,10-mechanistic pathway that avoids the formation of (3R)-NDP has also been suggested. In this alternative scenario, the final cadinenyl cation is proposed to be formed through the intermediacy of trans, trans-germacradienyl cation and germacrene D. In addition, an alternative 1,6-ring closure mechanism via the bisabolyl cation has previously been envisioned. We report here a detailed investigation of the catalytic mechanism of DCS using a variety of mechanistic probes including, among others, deuterated and fluorinated FDPs. Farnesyl diphosphate analogues with fluorine at C2 and C10 acted as inhibitors of DCS, but intriguingly, after prolonged overnight incubations, they yielded 2F-germacrene(s) and a 10F-humulene, respectively. The observed 1,10-, and to a lesser extent, 1,11-cyclization activity of DCS with these fluorinated substrates is consistent with the postulated macrocyclization mechanism(s) en route to (+)-δ-cadinene. On the other hand, mechanistic results from incubations of DCS with 6F-FPP, (2Z,6E)-FDP, neryl diphosphate, 6,7-dihydro-FDP, and NDP seem to be in better agreement with the potential involvement of the alternative biosynthetic 1,6-ring closure pathway. In particular, the strong inhibition of DCS by 6F-FDP, coupled to the exclusive bisabolyl- and terpinyl-derived product profiles observed for the DCS-catalyzed turnover of (2Z,6E)-farnesyl and neryl diphosphates, suggested the intermediacy of α-bisabolyl cation. DCS incubations with enantiomerically pure [1-(2)H(1)](1R)-FDP revealed that the putative bisabolyl-derived 1,6-pathway proceeds through (3R)-nerolidyl diphosphate (NDP), is consistent with previous deuterium-labeling studies, and accounts for the cis stereochemistry characteristic of cadinenyl-derived sesquiterpenes. While the results reported here do not unambiguously rule in favor of 1,6- or 1,10-cyclization, they demonstrate the mechanistic versatility inherent to DCS and highlight the possible existence of multiple mechanistic pathways.

Nonradioactive assay for detecting isoprenyl diphosphate synthase activity in crude plant extracts using liquid chromatography coupled with tandem mass spectrometry

Terpenoids form the largest class of plant metabolites involved in primary and secondary metabolism. Isoprenyl diphosphate synthases (IDSs) catalyze the condensation of the C5 terpenoid building blocks, isopentenyl diphosphate and dimethylallyl diphosphate, to form geranyl diphosphate (C10), farnesyl diphosphate (C15), and geranylgeranyl diphosphate (C20). These branch point reactions control the flow of metabolites that act as precursors to each of the major terpene classes—monoterpenes, sequiterpenes, and diterpenes, respectively. Thus accurate and easily performed assays of IDS enzyme activity are critical to increase our knowledge about the regulation of terpene biosynthesis. Here we describe a new and sensitive nonradioactive method for carrying out IDS assays using liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) to detect the short-chain prenyl diphosphate products directly without dephosphorylation. Furthermore, we were able to separate cisoid and transoid isomers of both C10 enzyme products (geranyl diphosphate and neryl diphosphate) and three C15 products [(E,E)-, (Z,E)-, and (Z,Z)-farnesyl diphosphate]. By applying the method to crude protein extracts from various organs of Arabidopsis thaliana, Nicotiana attenuata, Populus trichocarpa, and Picea abies, we could determine their IDS activity in a reproducible fashion.

Molecular Cloning and Characterization of a Linalool Synthase from Lemon Myrtle

Using a homology-based PCR strategy, we identified a cDNA with sequence similarity to linalool synthase from lemon myrtle. Functional expression of the cDNA (designated BcLS) gene in Escherichia coli yielded an active enzyme capable of catalyzing the conversion of geranyl diphosphate to (-)-linalool, i.e., an acyclic monoterpene alcohol, and to lesser amounts of cyclic monoterpenes. The kinetic parameters of BcLS were similar to those of synthases producing cyclic monoterpenes. PCR analysis revealed that the BcLS gene transcript was ubiquitously expressed in lemon myrtle and was upregulated in response to jasmonic acid treatment. Although the physiological role of neryl diphosphate (NPP) dependency of BcLS remains unclear, the cyclization activity of BcLS was enhanced when NPP was used as substrate, resulting in predominant production of cyclic monoterpenes. These findings indicate that BcLS has novel specificity and kinetic parameters, but its physiological responses to stresses such as insect damage appear to be similar to known linalool synthases.

Monoterpenes in the glandular trichomes of tomato are synthesized from a neryl diphosphate precursor rather than geranyl diphosphate

We identified a cis-prenyltransferase gene, neryl diphosphate synthase 1 (NDPS1), that is expressed in cultivated tomato (Solanum lycopersicum) cultivar M82 type VI glandular trichomes and encodes an enzyme that catalyzes the formation of neryl diphosphate from isopentenyl diphosphate and dimethylallyl diphosphate. mRNA for a terpene synthase gene, phellandrene synthase 1 (PHS1), was also identified in these glands. It encodes an enzyme that uses neryl diphosphate to produce beta-phellandrene as the major product as well as a variety of other monoterpenes. The profile of monoterpenes produced by PHS1 is identical with the monoterpenes found in type VI glands. PHS1 and NDPS1 map to chromosome 8, and the presence of a segment of chromosome 8 derived from Solanum pennellii LA0716 causes conversion from the M82 gland monoterpene pattern to that characteristic of LA0716 plants. The data indicate that, contrary to the textbook view of geranyl diphosphate as the "universal" substrate of monoterpene synthases, in tomato glands neryl diphosphate serves as a precursor for the synthesis of monoterpenes.

Farnesyl Diphosphate Synthase: The Art of Compromise between Substrate Selectivity and Stereoselectivity

Farnesyl diphosphate (FPP) synthase catalyzes the consecutive head-to-tail condensations of isopentenyl diphosphate (IPP, C5) with dimethylallyl diphosphate (DMAPP, C5) and geranyl diphosphate (GPP, C10) to give (E,E)-FPP (C15). The enzyme belongs to a genetically distinct family of chain elongation enzymes that install E-double bonds during each addition of a five-carbon isoprene unit. Analysis of the C10 and C15 products from incubations with avian FPP synthase reveals that small amounts of neryl diphosphate (Z-C10) and (Z,E)-FPP are formed along with the E-isomers during the C5 --> C10 and C10 --> C15 reactions. Similar results were obtained for FPP synthase from Escherichia coli, Artemisia tridentata (sage brush), Pyrococcus furiosus, and Methanobacter thermautotrophicus and for GPP and FPP synthesized in vivo by E. coli FPP synthase. When (R)-[2-2H]IPP was a substrate for chain elongation, no deuterium was found in the chain elongation products. In contrast, the deuterium in (S)-[2-2H]IPP was incorporated into all of the products. Thus, the pro-R hydrogen at C2 of IPP is lost when the E- and Z-double bond isomers are formed. The synthesis of Z-double bond isomers by FPP synthase during chain elongation is unexpected for a highly evolved enzyme and probably reflects a compromise between optimizing double bond stereoselectivity and the need to exclude DMAPP from the IPP binding site.

Decaprenyl diphosphate synthesis in Mycobacterium tuberculosis.

Z-prenyl diphosphate synthases catalyze the sequential condensation of isopentenyl diphosphate with allylic diphosphates to synthesize polyprenyl diphosphates. In mycobacteria, these are precursors of decaprenyl phosphate, a molecule which plays a central role in the biosynthesis of essential mycobacterial cell wall components, such as the mycolyl-arabinogalactan-peptidoglycan complex and lipoarabinomannan. Recently, it was demonstrated that open reading frame Rv2361c of the Mycobacterium tuberculosis H37Rv genome encodes a unique prenyl diphosphate synthase (M. C. Schulbach, P. J. Brennan, and D. C. Crick, J. Biol. Chem. 275:22876-22881, 2000). We have now purified the enzyme to near homogeneity by using an Escherichia coli expression system and have shown that the product of this enzyme is decaprenyl diphosphate. Rv2361c has an absolute requirement for divalent cations and an optimal pH range of 7.5 to 8.5, and the activity is stimulated by both detergent and dithiothreitol. The enzyme catalyzes the addition of isopentenyl diphosphate to geranyl diphosphate, neryl diphosphate, omega,E,E-farnesyl diphosphate, omega,E,Z-farnesyl diphosphate, or omega,E,E,E-geranylgeranyl diphosphate, with Km values for the allylic substrates of 490, 29, 84, 290, and 40 microM, respectively. The Km value for isopentenyl diphosphate is 89 microM. The catalytic efficiency is greatest when omega,E,Z-farnesyl diphosphate is used as the allylic acceptor, suggesting that this is the natural substrate in vivo, a conclusion that is supported by previous structural studies of decaprenyl phosphoryl mannose isolated from M. tuberculosis. This is the first report of a bacterial Z-prenyl diphosphate synthase that preferentially utilizes an allylic diphosphate primer having the alpha-isoprene unit in the Z configuration, indicating that Rv1086 (omega,E,Z-farnesyl diphosphate synthase) and Rv2361c act sequentially in the biosynthetic pathway that leads to the formation of decaprenyl phosphate in M. tuberculosis.

Biosynthesis of Marine Natural Products: Isolation and Characterization of a Myrcene Synthase from Cultured Tissues of the Marine Red Alga Ochtodes secundiramea

The acyclic monoterpene myrcene is the likely progenitor of the unusual cytotoxic halogenated monoterpenes that are found in marine algae and that function as feeding deterrents to herbivores. Myrcene synthase was isolated from suspension cultures of the marine red alga Ochtodes secundiramea, representing the first enzyme of this type from a marine organism. The algal myrcene synthase produces exclusively myrcene from the natural substrate geranyl diphosphate (GDP), utilizes Mg+2 as the required divalent metal ion cofactor, has a molecular mass of about 69 kDa, and exhibits a pH optimum near 7.2. These features are similar to those of monoterpene synthases from terrestrial organisms. When incubated with neryl diphosphate (the cis-isomer of GDP), the O. secundiramea myrcene synthase produces the cyclic monoterpene limonene, whereas incubation with (±)linalyl diphosphate (the tertiary allylic isomer of geranyl diphosphate) yields both acyclic and cyclic monoterpenes. These results suggest that the enzyme is incapable of isomerizing geranyl diphosphate to linalyl diphosphate, a feature common to all monoterpene cyclases from terrestrial sources. The limited catalytic capability of the myrcene synthase may reflect the ancient evolutionary origin of the producing organism. The ability to assay this enzyme in cultured algae, grown under strictly defined conditions, provides an unparalleled opportunity to delineate factors eliciting the biosynthesis of this class of secondary metabolites, to investigate the metabolic pathway leading to the halogenated monoterpenes, and to determine their role in the chemical ecology of marine algae.

CHARACTERIZATION OF A MYRCENE SYNTHASE FROM SUSPENSION CULTURES OF THE MARINE RED ALGA OCHTODES SECUNDIRAMEA

Wise, M. L.1, Rorrer, G. L.2, Polzin, J. J.2, Croteau, R. B.1 1Institute of Biological Chemistry, Washington State University, Pullman, WA. 99164 USA; 2 Department of Chemical Engineering, Oregon State University, Corvallis, OR. 96331USAA monoterpene synthase from suspension cultures of the marine red alga Ochtodes secundiramea is shown to biosynthesize myrcene from geranyl diphosphate (GPP) using cell free extracts. This is the first in vitro characterization of a monoterpene synthase from a marine organism. Myrcene is the likely progenitor of the unusual halogenated monoterpenes characteristic of this marine alga and, as such, represents a key step in the biosynthetic pathway. Based on mechanistic considerations from reaction with the biologically relevant substrate GPP, as well as neryl diphosphate (the cis isomer of GPP) and linalyl diphosphate (LPP), the enzyme appears incapable of catalyzing the isomerization of GPP to LPP, a mechanistic feature of most terrestrial monoterpene synthases, perhaps reflecting its evolutionarily ancient origin. The ability to assay and quantitatively monitor the expression of this enzyme in suspension cultures, under strictly defined growth conditions, presents an unparalleled opportunity to delineate, at the molecular level, factors eliciting the biosynthesis of this class of secondary metabolites, to evaluate the metabolic pathway leading to halogenated monoterpenes and to investigate their role in the chemical ecology of marine algae.