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The mevalonate pathway plays an important role in providing the cell with a number of essential precursors for the synthesis of biomass constituents. With respect to their chemical structure, the metabolites of this pathway can be divided into two groups: acyl esters [acetoacetyl CoA, acetyl CoA, hydroxymethylglutaryl (HMG) CoA] and phosphorylated metabolites (isopentenyl pyrophosphate, dimethylallyl pyrophosphate, geranyl pyrophosphate, farnesyl pyrophosphate). In this study, we developed a method for the precise analysis of the intracellular concentration of acetoacetyl CoA, acetyl CoA and HMG CoA; and we used this method for quantification of these metabolites in Saccharomyces cerevisiae, both during batch growth on glucose and on galactose and in glucose-limited chemostat cultures operated at three different dilution rates. The level of the metabolites changed depending on the growth phase/specific growth rate and the carbon source, in a way which indicated that the synthesis of acetoacetyl CoA and HMG CoA is subject to glucose repression. In the glucose batch, acetyl CoA accumulated during the growth on glucose and, just after glucose depletion, HMG CoA and acetoacetyl CoA started to accumulate during the growth on ethanol. In the galactose batch, HMG CoA accumulated during the growth on galactose and a high level was maintained into the ethanol growth phase; and the levels of acetyl CoA and HMG CoA were more than two-fold higher in the galactose batch than in the glucose batch.

J. Inst. Brew. 110(3), 213–219, 2004 Geraniol is the main monoterpenol present in Gewurtztraminer white grapes and in some red aromatic grapes from northern Italy. During fermentation, the geraniol concentration showed a dramatic drop and a small amount was transformed into linalool and -terpineol by an acid catalysed chemical reaction. Yeast are responsible for changing most of the geraniol to citronellol through enzymatic reactions. The final aroma of wine is due mainly to both the untransformed geraniol and the formed citronellol. In this study, competition between the geraniol-citronellol transformation and another metabolic process, in which according to our hypothesis sterol biosynthesis is involved, was investigated. Geraniol, in the active form of geranyl pyrophosphate, is one of intermediates in that metabolic pathway. The addition of ergosterol, one of final products of sterol biosynthesis, to the fermentation medium, appeared to reduce the amount of geraniol involved in this metabolic pathway; as a result geraniol-citronellol transformation was favoured. The result was a higher production of citronellol with the same geraniol consumption. The relationships between oxygen and regulation of the metabolic pathways involving geraniol were also studied. Results showed that aerobic conditions could improve citronellol production. Finally, it was observed that metabolism of geraniol also depended on the physiological state of the yeast during fermentation.

Farnesyl pyrophosphate synthetase (FPPS) synthesizes farnesyl pyrophosphate through successive condensations of isopentyl pyrophosphate with dimethylallyl pyrophosphate and geranyl pyrophosphate. Nitrogen-containing bisphosphonate drugs used to treat osteoclast-mediated bone resorption and tumor-induced hypercalcemia are potent inhibitors of the enzyme. Here we present crystal structures of substrate and bisphosphonate complexes of FPPS. The structures reveal how enzyme conformational changes organize conserved active site residues to exploit metal-induced ionization and substrate positioning for catalysis. The structures further demonstrate how nitrogen-containing bisphosphonates mimic a carbocation intermediate to inhibit the enzyme. Together, these FPPS complexes provide a structural template for the design of novel inhibitors that may prove useful for the treatment of osteoporosis and other clinical indications including cancer.

Cholesterol metabolites play a several critical roles in regulating cell growth and function. 3-Hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase, the rate-limiting enzyme for this pathway, is down regulated by feedback mechanisms due to increased levels of cholesterol and its premetabolites. Several HMG-CoA metabolites, such as farnesyl pyrophosphate and geranyl pyrophosphate are implicated in oncogene activation and tumorigenesis. Recent studies suggest that inhibition of HMG-CoA reductase by specific inhibitors or by naturally-occurring phytochemicals, such as farnesol or squalene can modulate tumor cell growth. Thus, in this study, we have assessed the chemopreventive efficacy of farnesol and lanosterol on azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) in rats. In addition, we measured the effect of farnesol and lanosterol on serum high denisity lipoprotein (HDL) and cholesterol levels in the rats. Seven-week-old male F344 rats were fed the control diet (modified AIN-76A) or experimental diets containing I or 2% lanosterol or 1.5% farnesol. One week later, all animals except those in vehicle (normal saline)-treatment groups were s.c. injected with AOM (15 mg/kg body weight, once weekly for 2 weeks). At 16 weeks of age, all rats were killed, colons were evaluated for ACF and serum was assayed for HDL and cholesterol levels. Administration of dietary farnesol significantly inhibited ACF formation by about 34% (P0.001) and reduced crypt multiplicity by about 44% (P0.0001). Also, administration of lanosterol at dose levels of I or 2 % in the diet significantly suppressed AOM-induced colonic ACF as well as multicrypt foci formation. (P0.01-0.001). Further, farnesol at 1.5% and lanosterol at 1% did not show any significant effect on serum HDL nor on total cholesterol levels. However, lanosterol at 2% significantly increased serum HDL (P0.05) and cholesterol (P0.01) levels. That farnesol and lanosterol significantly suppress colonic ACF formation and crypt multiplicity strengthens the hypothesis that these agents possess chemopreventive activity against colon carcinogenesis.

To study the effect of salt stress on sterol biosynthesis through the activity of 3-hydroxy-3-methylglutaryl CoA reductase (HMG R) in halophilic Hortaea werneckii.

Farnesyl pyrophosphate (FPP) serves as a common substrate for many prenyltransferases involved in the biosynthesis of isoprenoid compounds. Undecaprenyl pyrophosphate synthase (UPPs) catalyzes the chain elongation of FPP to C(55) undecaprenyl pyrophosphate (UPP) which acts as a lipid carrier in bacterial peptidoglycan synthesis. In this study, 7-(2,6-dimethyl-8-diphospho-2,6-octadienyloxy)-8-methyl-4-trifluoromethyl-chromen-2-one geranyl pyrophosphate, a fluorescent analogue of FPP, was prepared and utilized to study ligand interactions with E. coli UPPs. This compound displays an absorbance maximum at 336 nm and emission maximum at 460 nm without interference from protein autofluorescence. It is a competitive inhibitor with respect to FPP (K(i) = 0.57 microM) and also serves as an alternative substrate (K(m) = 0.69 microM and k(cat) = 0.02 s(-)(1)), but mainly reacts with one isopentenyl pyrophosphate (IPP) probably due to unfavorable product translocation. Fluorescence intensity of this compound is reduced when bound to the enzyme (1:1 stoichiometry), and is recovered by FPP replacement. Using stopped-flow apparatus, the interaction of enzyme with the compound was measured (k(on) = 55.3 microM(-)(1) s(-)(1) and k(off) = 31.6 s(-)(1)). The product dissociation rate constant (0.5 s(-)(1)) determined from the competition experiments is consistent with our previous prediction from kinetic simulation. Unlike several other prenyltransferase reactions in which FPP dissociates slowly, UPPs binds FPP in a rapid equilibrium manner with a fast release rate constant of 30 s(-)(1). The fluorescent analogue of FPP presented here may provide a tool to investigate the ligand interactions for a broad class of FPP-binding proteins.

The possibility that ubiquinone biosynthesis is present in rat liver peroxisomes was investigated. The specific activity of trans -prenyltransferase was 30% that of microsomes, with a pH optimum of around 8. trans -Geranyl pyrophosphate was required as a substrate and maximum activity was achieved with Mn 2+ . Several detergents specifically inactivated the peroxisomal enzyme. The peroxisomal transferase is present in the luminal soluble contents, in contrast to the microsomal enzyme which is a membrane component. The treatment of rats with a number of drugs has demonstrated that the activities in the two organelles are subjected to separate regulation. Nonaprenyl-4-hydroxybenzoate transferase has about the same specific activity in peroxisomes as in microsomes and like the transferase activity, its regulation differs from the microsomal enzyme. The results demonstrate that peroxisomes are involved in ubiquinone biosynthesis, and at least two enzymes of the biosynthetic sequence are present in this organelle.

ATP-binding cassette transporter A1 (ABCA1) mediates an active efflux of cholesterol and phospholipids and is mutated in patients with Tangier disease. Expression of ABCA1 may be increased by certain oxysterols such as 22(R)-hydroxycholesterol via activation of the nuclear hormone receptor liver X receptor (LXR). In searching for potential modulators of ABCA1 expression, we have studied the effects of various mevalonate metabolites on the expression of ABCA1 in two human cell lines, THP-1 and Caco-2 cells. Most of the tested metabolites, including mevalonate, geranyl pyrophosphate, farnesyl pyrophosphate, and ubiquinone, failed to significantly change the expression levels of ABCA1. However, treatment with geranylgeranyl pyrophosphate resulted in a dose- and time-dependent reduction of ABCA1 expression. Geranylgeranyl pyrophosphate appears to reduce ABCA1 expression via two different mechanisms. One of these mechanisms is by acting directly as an antagonist of LXR since it reduces the interaction between LXR alpha or -beta with nuclear coactivator SRC-1. Another mechanism appears to involve activation of the Rho GTP-binding proteins since treatment of Caco-2 cells with inhibitors of geranylgeranyl transferase or the Rho proteins significantly increased the expression and promoter activity of ABCA1. Further studies showed that mutations in the DR4 element of the ABCA1 promoter completely eliminate the inducible activities of these inhibitors. These data indicate that activation of the Rho proteins may change the activation status of LXR.

Monoterpenes are compounds found in the essential oils extracted from many plants, including fruits, vegetables, spices and herbs. These compounds contribute to the flavor and aroma of plant from which they are extracted. Monoterpenes are acyclic, monocyclic, or bicyclic C30 compounds synthesized by monoterpene synthases using geranyl pyrophosphate (GPP) as substrate. GPP is also the precursor in the synthesis of farnesyl pyrophosphate (FPP) and geranyl-geranyl pyrophosphate (GGPP), two important compounds in cell metabolism of animals, plants and yeast. Monoterpene cyclases produce cyclic monoterpenes through a multistep mechanism involving a universal intermediate, a terpinyl cation which can be transformed to several compounds. Experimental studies, using animal cancer models, have demonstrated that some monoterpenes possess anticarcinogenic properties, acting at different cellular and molecular levels. From these discoveries it seems clear that monoterpenes could be considered as effective, nontoxic dietary antitumorigenic agents that hold promise as a novel class of anticancer drugs.

4S-(-)-limonene is the major monoterpene accumulated by the liverwort Ricciocarpos natans, and a cell-free extract from this nonvascular plant cultured in vitro catalyzes the cyclization of geranyl diphosphate to limonene. The time course of limonene synthase activity parallels cultured growth but shows a maximum in specific activity in the lag phase following transfer to fresh medium. The operationally soluble enzyme was partially purified by combination of anion-exchange and hydroxylapatite chromatography. The limonene synthase from R. natans possesses a molecular weight of about 51,000, exhibits a pH optimum at 6.5, a pI at 5.3, and a requirement for either Mg2+ or Mn2+ as cofactor, and appears to employ 3S-linalyl disphosphate as a bound intermediate in the cyclization of the geranyl substrate (Km approximately 1.25 microM). In stereochemistry of the coupled isomerization-cyclization reaction and in general properties, the limonene synthase from this bryophyte resembles the corresponding monoterpene cyclases from gymnosperm and angiosperm species.

The focus of studies of diet/cancer interrelationships is shifting from the roles nutrients play to the chemopreventive actions of plant-derived, non-nutrient dietary constituents (indoles, coumarins, phthalides, organosulfur compounds, phytoestrogens, isothiocy-anates, bioflavonoids and protease inhibitors). Our interest in this aspect of diet/health relationships has roots in findings that products of plant mevalonate pathways both suppress hepatic mevalonate synthesis with a concomitant lowering of serum LDL cholesterol level and suppress the growth of chemically-initiated and transplanted tumors.1–4 Some of the more effective end products derived from the sequential mevalonate pathway intermediates, geranyl pyrophosphate, farnesyl pyrophosphate and geranylgeranyl pyrophosphate, are listed on Figure 1.1, 2 The pathway provides essential dietary components, specifically phylloquinone, β-carotene and a-tocopherol. The monoterpenes and sesquiterpenes have no nutritional impact. Never the less each of the products listed on Figure 1 has been shown to have an impact on HMG CoA reductase activity, blood cholesterol level and tumor cell proliferation.1–4

Limonene and related monoterpenes display compelling anticarcinogenic activity. The mechanism(s) that underlie this activity is/are as yet unknown. One attractive possibility is that the monoterpenes interact with the RAS signal transduction pathway. The monoterpenes have been shown to impair incorporation of mevalonic acid-derived isoprene compounds, that is farnesyl pyrophosphate, into RAS and RAS-related proteins. As farnesylation is critical for RAS’s membrane localization and function, the isoprenylation pathways have received attention as potential targets of anti-RAS pharmacological maneuvers. We have expanded on prior studies and demonstrate that one of limonene’s metabolic derivatives, perillyl alcohol, decreases the levels of antigenic RAS in the human-derived myeloid THP-1 and lymphoid RPMI-8402 leukemia cell lines. Both limonene and perillyl alcohol decrease levels of 35[S]-methionine labeled RAS proteins in cells that have been pulsed with radiolabeled methionine for four hours. In contrast, lovastatin, which inhibits hydroxymethylglutaryl coenzyme A reductase and thus depletes cells of farnesyl pyrophosphate, does not diminish levels of total antigenic RAS but rather results in a shift in the RAS protein; levels of farnesylated RAS decrease whereas levels of unmodified/unfarnesylated RAS increase. As limonene and perillyl alcohol do not induce such a shift we conclude that these monoterpenes decrease farnesylated RAS protein levels by a mechanism that is clearly distinct from that of either depleting cells of farnesyl pyrophosphate or inhibiting the enzyme farnesyl protein transferase that catalyzes the posttranslational farnesylation of RAS. These findings are discussed with respect to implications for the monoterpenes to alter RAS protein synthesis and degradation. The results of these studies will likely impact the inclusion of the monoterpenes in clinical anticancer trials.

Diets supplemented (1 mmol/kg) with thymol, carvacrol, and beta-ionone significantly decreased the serum cholesterol levels of cockerels. These mevalonate-derived end products of plant secondary metabolism (isoprenoids) had no impact on two cytosolic prenyl alcohol (and ethanol) dehydrogenase activities; each treatment increased microsomal geranyl pyrophosphate pyrophosphatase activity by greater than twofold. The structural diversity of the isoprenoids which suppress cholesterol synthesis may be reconciled by their ability to increase pyrophosphatase activity, thus leading to the production of the endogenous, post-transcriptional regulator of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity.

Protein geranylgeranyltransferase-I (PGGT-I) catalyzes the transfer of the 20-carbon prenyl group from geranylgeranyl pyrophosphate to the cysteine residue near the C-termini of a variety of eukaryotic proteins. Kinetic analysis of homogenous PGGT-I from bovine brain reveals that the reaction follows a sequential pathway in which either prenyl donor or acceptor can bind first to the enzyme and that the reaction operates at steady-state rather than at rapid equilibrium. Substrate inhibition by prenyl acceptor but not by prenyl donor suggests that geranylgeranyl pyrophosphate binding first to free enzyme is the kinetically preferred pathway. This is supported by isotope trapping experiments which show that the ternary complex goes on to products faster than the release of geranylgeranyl pyrophosphate from the complex. The KM for the interaction of geranylgeranyl pyrophosphate with PGGT-I is markedly affected by the structure of the prenyl acceptor bound to the enzyme. A detailed analysis of the substrate specificity of PGGT-I reveals that peptides which contain a C-terminal leucine are preferred (kcat/KM = 1-5 x 10(5) M-1 s-1) to those that end in serine (kcat/KM = 2-4 x 10(3) M-1 s-1) or phenylalanine (kcat/KM = 0.5 x 10(3) M-1 s-1). PGGT-I also catalyzes the farnesylation of peptides that have a C-terminal leucine; kcat for farnesylation and KM for farnesyl pyrophosphate are similar to those for geranylgeranylation, but the KM for the peptide is 30-fold higher. Geranyl pyrophosphate is utilized by PGGT-I but is a poor substrate. Optimal activity of PGGT-I is obtained in the presence of micromolar amounts of Zn2+ and mM amounts of Mg2+. Mn2+ or Cd2+ but not Co2+ can substitute for Zn2+ and for Mg2+. Metals are not required for tight-binding of geranylgeranyl pyrophosphate to PGGT-I, and the measured dissociation equilibrium constant for this binary complex is 16 nM. Photoaffinity analogues of geranylgeranyl pyrophosphate and farnesyl pyrophosphate were prepared and shown to exclusively label the beta-subunit. The implication of the results for the substrate specificity of protein prenylation in cells is briefly discussed.

The three pinene synthases (cyclases) from common sage ( Salvia officinalis ) catalyze the conversion of geranyl pyrophosphate to the bicyclic olefins (+)-α-pinene and (+)-camphene (cyclase I), (−)-α-pinene, (−)-β-pinene, and (−)-camphene (cyclase II), and (+)-α-pinene and (+)-β-pinene (cyclase III), in addition to smaller amounts of monocyclic and acyclic monoterpene olefins. [1- 3 H,4- 2 H 2 ]- and [10- 2 H 2 ]-geranyl pyrophosphates were prepared and used in conjunction with 1- 3 H- and 1- 3 H,10- 2 H 3 -labeled geranyl precursors to examine the isotope effects attending the C4- and C10-deprotonation steps in the enzymatic synthesis of the pinenes. The observation of isotopically sensitive branching within each set of stereochemically related bicyclic olefins confirmed that each product set was synthesized by the respective pinene synthase by partitioning of common carbocationic intermediates along different reaction channels at the active site. The changes in product distribution resulting from deuterium substitution at C4 and C10 of the substrate were used to determine kinetic isotope effects (KIEs) for the terminating deprotonations; these observed KIEs represent the lower limits of the intrinsic isotope effects. The intramolecular isotope effects for the methyl-methylene elimination in β-pinene formation by cyclases II and III were also evaluated with [10- 2 H 2 ]geranyl pyrophosphate as substrate and by MS analysis of the olefin products. The intramolecular KIEs ( k H / k D = 3.0 and 3.5) were significantly higher than the observed KIEs determined from product ratios ( k H / k D = 1.7 and 2.6) since the former involves considerably less masking of the intrinsic isotope effects.

The genetic basis of plant monoterpene composition has been the subject of detailed investigation, most notably an extensive series of crossing experiments performed on species of mints (Mentha) by M.J. Murray and his coworkers. This research, which began in the 1950s, identified a number of genes regulating the production of the principal essential oil components in Mentha.1,2 More recently, Croteau and associates have examined the biosynthesis of the major monoterpenes of the commercial Mentha species (peppermint, native spearmint and Scotch spearmint). Using both in vivo and in vitro experimental systems,3-5 the sequence of intermediates has been established and the relevant enzymes have been isolated and characterized. In the last year, a gene encoding a key enzyme of the pathway has been isolated, sequenced and functionally expressed in a bacterial host.6

Geranylgeranyl pyrophosphate (GGPP) synthase from Erwinia uredovora was overexpressed in Escherichia coli and purified to homogeneity from solubilized inclusion bodies. In this protein the first 13 N-terminal amino acids were replaced by 16 other amino acids resulting from the cloning vector pUC 18. Nevertheless, the enzyme showed activity after purification which could be stimulated sixfold by appropriate activation conditions. The homogeneous enzyme was used to study substrate and product specificity as well as to determine K m values for isopentenyl pyrophosphate, dimethylallyl pyrophosphate (DMAPP), geranyl pyrophosphate (GPP), and farnesyl pyrophosphate (FPP). Reaction rates and K m values indicate that FPP and GPP are the genuine allylic substrates for GGPP synthase, but not DMAPP. Independent of the allylic substrate employed, GGPP was the only reaction product of the enzymatic reaction.

The biosynthesis of several monoterpenes from the acyclic precursor geranyl pyrophosphate requires the migration of positive charge from the isopropyl side chain into the cyclohexenoid ring of the universal alpha-terpinyl cation intermediate of the reaction. The hydride shifts responsible for charge migration in the formation of three p-menthane olefin isomers were examined in a range of plant species: alpha-terpinene in American wormseed, gamma-terpinene in thyme, and (-)-beta-phellandrene in lodgepole pine. The experimental approach utilized soluble cell-free enzyme systems and specifically labeled geranyl substrates, with the determination of the labeling patterns in the resulting cyclic products. The results were consistent with stereoelectronic features of the cyclization and support the general model for monoterpene formation.

We report here on the characterization of a cDNA library from seeds of Jatropha curcas L. at three stages of fruit maturation before yellowing. We sequenced a total of 2200 clones and obtained a set of 931 non-redundant sequences (unigenes) after trimming and quality control, ie, 140 contigs and 791 singlets with PHRED quality $10. We found low levels of sequence redundancy and extensive metabolic coverage by homology comparison to GO. After comparison of 5841 non-redundant ESTs from a total of 13193 reads from GenBank with KEGG, we identified tags with nucleotide variations among J. curcas accessions for genes of fatty acid, terpene, alkaloid, quinone and hormone pathways of biosynthesis. More specifically, the expression level of four genes (palmitoyl-acyl carrier protein thioesterase, 3-ketoacyl-CoA thiolase B, lysophosphatidic acid acyltransferase and geranyl pyrophosphate synthase) measured by real- time PCR proved to be significantly different between leaves and fruits. Since the nucleotide polymorphism of these tags is associated to higher level of gene expression in fruits compared to leaves, we propose this approach to speed up the search for quantitative traits in selective breeding of J. curcas. We also discuss its potential utility for the selective breeding of economically important traits in J. curcas. Diponível em:.Acesso em: 03 nov. 2010.

(4 S )-Limonene synthase, isolated from glandular trichome secretory cell preparations of Mentha × piperita (peppermint) leaves, catalyzes the metal ion-dependent cyclization of geranyl pyrophosphate, via 3 S -linalyl pyrophosphate, to (−)-(4 S )-limonene as the principal product. Treatment of this terpene cyclase with the histidine-directed reagent diethyl pyrocarbonate at a concentration of 0.25 m m resulted in 50% loss of enzyme activity, and this activity could be completely restored by treatment of the preparation with 5 m m hydroxylamine. Inhibition with diethyl pyrocarbonate was distinguished from inhibition with thiol-directed reagents by protection studies with histidine and cysteine carried out at varying pH. Inactivation of the cyclase by dye-sensitized photooxidation in the presence of rose bengal gave further indication of the presence of a readily modified histidine residue. Protection of the enzyme against inhibition with diethyl pyrocarbonate was afforded by the substrate geranyl pyrophosphate in the presence of Mn 2+ , and by the sulfonium ion analog of the linalyl carbocation intermediate of the reaction in the presence of inorganic pyrophosphate plus Mn 2+ , suggesting that an essential histidine residue is located at or near the active site. Similar studies on the inhibition of other monoterpene and sesquiterpene cyclases with diethyl pyrocarbonate suggest that a histidine residue (or residues) may play an important role in catalysis by this class of enzymes.

(4S)-Limonene synthase, a monoterpene cyclase isolated from the secretory cells of the glandular trichomes of Mentha x piperita (peppermint), catalyzes the cyclization of geranyl pyrophosphate to (4S)-limonene, a key intermediate in the biosynthesis of p-menthane monoterpenes in Mentha species. The enzyme synthesizes principally (-)-(4S)-limonene (greater than 94% of the total products), plus several other monoterpene olefins. The general properties of (4S)-limonene synthase resemble those of other monoterpene cyclases. The enzyme shows a pH optimum near 6.7, an isoelectric point of 4.35, and requires a divalent metal ion for catalysis, either Mg2+ or Mn2+, with Mn2+ preferred. The Km value measured for geranyl pyrophosphate was 1.8 microM. The activity of (4S)-limonene synthase was inhibited by sodium phosphate, sodium pyrophosphate, and reagents directed against the amino acids cysteine, methionine, and histidine. In the presence of Mn2+, geranyl pyrophosphate protected against cysteine-directed inhibition, suggesting that at least one cysteine residue is located at or near the active site. Experiments with alternate substrates and substrate analogs confirmed many elements of the proposed reaction mechanism, including the binding of geranyl pyrophosphate in the form of a complex with the divalent metal ion, the preliminary isomerization of geranyl pyrophosphate to linalyl pyrophosphate (a bound intermediate capable of cyclization), and the participation of a series of carbocation:pyrophosphate anion pairs in the reaction sequence.

A major wound response in grand fir (Abies grandis) sapling stems is the rapid increase in monoterpene production at the site of injury. Monoterpene cyclases (synthases) catalyze the formation of monoterpenes from geranyl pyrophosphate, and total cyclase activity increases markedly on wounding. At least six distinct cyclases, producing different monoterpene products, have been isolated from wounded grand fir saplings and characterized. The predominant wound-inducible cyclase produces both α- and β-pinene. This pinene cyclase was purified, and polyclonal antibodies were generated in rabbits against the sodium dodecyl sulfate-denatured protein. The antibody preparation was found to crossreact by Western blotting with other grand fir monoterpene cyclases that produce different olefinic products, but not with monoterpene cyclases from related conifer species (Pinus contorta and P. ponderosa) or from angiosperms (Mentha piperita and M. spicata). The increase in monoterpene cyclase activity after wounding was closely correlated with the appearance of new cyclase protein as determined by immunoblotting. These results indicate that the wound-dependent increase in monoterpene cyclase activity is a consequence of de novo synthesis of cyclase protein.

Monoterpenes are the C10 members of the terpenoid family of natural products, and they diverge from higher isoprenoid biosynthesis at the level of geranyl pyrophosphate. γ-Terpinene synthase and limonene synthase catalyze the divalent metal ion-dependent cyclization of geranyl pyrophosphate to cyclic monoterpenes of the p-menthane type; however, γ-terpinene synthase generates a diverse assortment of minor monoterpene co-products from geranyl pyrophosphate whereas limonene synthase produces a single product. The basic physical and chemical characteristics of these two monoterpene cyclases are described and compared to properties of other terpene cyclases.

Hyssop (Hyssopus officinalis) produces an essential oil containing the saturated bicyclic monoterpene ketones pinocamphone and isopinocamphone, with lesser amounts of myrtenol derivatives. A microsomal preparation from leaf epidermis oil glands of this species converts the parent olefin (-)-β -pinene to the allylic alcohol (+)-trans-pinocarveol that presumably gives rise to (-)-pinocamphone and (-)-isopinocamphone by subsequent oxidation and two stereochemical alternatives for reduction of the conjugated double bond. The same preparation catalyzes the hydroxylation of (-)-α-pinene to (-)-myrtenol at a slower rate. The pinene hydroxylase from the oil glands Hyssop has characteristics of a distinct cytochrome P-450 species, yet shares properties with other monoterpene olefin P-450 hydroxylases from the oil glands of several essential oil-producing plants. These results with pinane-type monoterpenes extend the observation that parent cyclic olefins are metabolized by a pathway involving allylic oxidation and conjugate reduction.

Secretory cells were isolated from the monoterpene-producing glandular trichomes (peltate form) of peppermint as clusters of eight cells each. These isolated structures were shown to be non-specifically permeable to low-molecular-weight, water-soluble cofactors and substrates. Short incubation periods with the polar dye Lucifer yellow iodoacetamide (Mr=660) resulted in a uniform staining of the cytoplasm, with exclusion of the dye from the vacuole. The molecular-weight exclusion limit for this permeability was shown to be less than approx. 1800, based on exclusion of fluorescein-conjugated dextran (Mr ∼ 1800). Intact secretory cell clusters very efficiently incorporated [(3)H]geranyl pyrophosphate into monoterpenes. The addition of exogenous cofactors and redox substrates affected the distribution of monoterpenes synthesized from [(3)H]geranyl pyrophosphate, demonstrating that the cell clusters were permeable to these compounds and that the levels of endogenous cofactors and redox substrates were depleted in the isolated cells. When provided with the appropriate cofactors, such as NADPH, NAD(+), ATP, ADP and coenzyme A, the isolated secretory cell clusters incorporated [(14)C]sucrose into monoterpenes, indicating that these structures are capable of the de-novo biosynthesis of monoterpenes from a primary carbon source, and that they maintain a high degree of metabolic competence in spite of their permeable nature.

Allylic addition—elimination reactions are widely used in the enzyme-catalysed formation of terpenoid metabolites. It has earlier been shown that the isoprenoid chain elongation reaction catalysed by farnesyl pyrophosphate synthase involving successive condensations of dimethylallyl pyrophosphate (DMAPP) and geranyl pyrophosphate (GPP) with isopentenyl pyrophosphate (IPP) corresponds to such an S E' reaction with net syn stereochemistry for the sequential electrophilic addition and proton elimination steps. Studies of the enzymic cyclization of farnesyl pyrophosphate (FPP) to pentalenene have now established the stereochemical course of two additional biological S E' reactions. Incubation of both (9R)-and (9S)-[9- 3 H, 4,8- 14 ] FPP with pentalenene synthase and analysis of the resulting labelled pentalenene has revealed that H-9 re of FPP becomes H-8 of pentalenene, while H-9 si undergoes net intramolecular transfer to the adjacent carbon, becoming H-l re (lH-loc) of pentalenene, as confirmed by subsequent experiments with [10- 2 H, 11- 13 C]FPP. These results correspond to net anti-stereochemistry in the intramolecular allylic addition—elimination reaction. The stereochemical course of a second S E < reaction has now been examined by analogous incubations of (4 S , 8 S )-[4,8- 3 H, 4,8- 14 C ]FPP and (4 R ,8R)-[4,8- 3 H, 4,8- 14 C ]FPP with pentalenene synthase. Determ ination of the distribution of label in the derived pentalenenes showed stereospecific loss of the original H-8 si proton. Analysis of the plausible conformation of the presumed reaction intermediates revealed that the stereochemical course of the latter reaction cannot properly be described as either syn or anti, since cyclization and subsequent double bond formation require significant internal motions to allow proper overlap of the scissile C—H bond with the developing carbocation.

The need for synthetically prepared flavour compounds arises from the fact that during the storage of foodstuffs a certain loss of flavour is inevitable. These losses can be compensated for by adding synthetically produced flavour compounds. Besides this, synthetic flavour compounds have the great advantage of being available in the required quantity and quality irrespective of crop variation and season.

Two prenyltransferases were located in cell cultures of Vitis vinifera. A geranyl pyrophosphate synthase (EC 2.5.1.1) was associated with plastid-like membranes whereas a farnesyl pyrophosphate synthase (EC 2.5.1.10) was found to be soluble.

Monoterpene cyclases catalyze the divalent metal ion-dependent conversion of the acyclic precursor geranyl pyrophosphate to a variety of monocyclic and bicyclic monoterpene skeletons. Examination of the kinetics of inhibition of cyclization by the pyrophosphate ester of (E)-4-[2-diazo-3-trifluoropropionyloxy]-3-methyl-2-buten-1-ol, a photolabile structural analog of the substrate, using a partially purified preparation of geranyl pyrophosphate:(+)-pinene cyclase and geranyl pyrophosphate:(+)-bornyl pyrophosphate cyclase from common sage (Salvia officinalis) evidenced (under dark conditions) strictly uncompetitive inhibition with Ki′ values of 3.2 and 4.7 μ m , respectively. These values are close to the corresponding Km values for the substrate with these two enzymes. This novel property of the substrate analog was also examined in the presence of two other inhibitors which bind to different domains of the cyclase active site (inorganic pyrophosphate and a sulfonium ion analog of a cyclic carbocationic intermediate of the reaction sequence (dimethyl-(4-methylcyclohex-3-en-1-yl)sulfonium iodide)) in order to address the mechanistic origins of the uncompetitive inhibition of cyclization. It was not possible, however, to rule out either an induced-fit mechanism or a sequential binding mechanism since the substrate is recognized by at least two binding domains and because direct examination of the effects of binding on cyclase conformation is currently not feasible. The substrate analog, although photoactive, did not give rise to light-dependent enzyme inactivation of greater magnitude than that obtained from ultraviolet light alone. The unusual behavior of the analog was attributed to intramolecular interaction of the electron-rich carbonyl group of the diazoester with the required divalent metal ion that is chelated by the pyrophosphate group. A photostable analog of geraniol that resembled the photoactive substrate analog in bearing a carbonyl function at C6 (6-oxo-3,7-dimethyloct-2(trans)en-1-ol) was prepared. Following foliar application to rapidly growing sage plants, this analog was seemingly activated to the corresponding pyrophosphate ester in vivo and selectively inhibited the activity of several cyclases in this tissue as evidenced by diminished production of the corresponding monoterpene end products.

The conversion of geranyl pyrophosphate to (+)-bornyl pyrophosphate and (+)-camphene is considered to proceed by the initial isomerization of the substrate to (−)-(3 R )-linalyl pyrophosphate and the subsequent cyclization of this bound intermediate. In the case of (−)-bornyl pyrophosphate and (−)-camphene, isomerization of the substrate to the (+)-(3 S )-linalyl intermediate precedes cyclization. The geranyl and linalyl precursors were shown to be mutually competitive substrates (inhibitors) of the relevant cyclization enzymes isolated from Salvia officinalis (sage) and Tanacetum vulgare (tansy) by the mixed substrate analysis method, demonstrating that isomerization and cyclization take place at the same active site. Incubation of partially purified enzyme preparations with (3 R )-[1 Z - 3 H]linalyl pyrophosphate plus [1- 14 C]geranyl pyrophosphate gave rise to double-labeled (+)-bornyl pyrophosphate and (+)-camphene, whereas incubation of enzyme preparations catalyzing the antipodal cyclizations with (3 S )-[1 Z - 3 H]-linalyl pyrophosphate plus [1- 14 C]geranyl pyrophosphate yielded double-labeled (−)-bornyl pyrophosphate and (−)-camphene. Each product was then transformed to the corresponding (+)- or (−)-camphor without change in the 3 H: 14 C isotope ratio, and the location of the tritium label was deduced in each case by stereoselective, base-catalyzed exchange of the exo -α-hydrogen of the derived ketone. The finding that the 1 Z - 3 H of the linalyl precursor was positioned at the endo -α-hydrogen of the corresponding camphor in all cases, coupled to the previously demonstrated retention of configuration at C1 of the geranyl substrate in these transformations, confirmed the syn -isomerization of geranyl pyrophosphate to linalyl pyrophosphate and the cyclization of the latter via the anti,endo - conformer. These relative stereochemical elements, in combination with the observed enantiospecificities of the enzymes for the linalyl intermediates, allows definition of the overall absolute stereochemistry of the coupled isomerization and cyclization of geranyl pyrophosphate to the antipodal camphane (bornane) and isocamphane monoterpenoids.

Publisher Summary Microorganisms and plants, which make carotenoids from acetylcoenzyme A via geranyl geranyl pyrophosphate, oxidize and cleave them in a variety of ways. Both central and excentric cleavage occur in these organisms. The best studied reaction in mammals is the oxidative cleavage of all-trans-β-carotene to two molecules of all-trans-retinal, a reaction catalyzed by β-carotenoid 15,15’-dioxygenase. The enzymatic cleavage of carotenoids to β-apocarotenals has not been unambiguously demonstrated in mammalian tissues, although chemical reactions of this kind occur at a slow rate in the presence of oxygen). This chapter focuses, therefore, on methods for measuring β-carotenoid 15,15’-dioxygenase. The chapter includes a brief treatment of excentric cleavage in microorganisms. Excentric cleavage clearly occurs in plants and microorganisms, as evidenced by the presence of β-apocarotenals and derivatives such as azafrin, bixin, and crocetin in nature.

Two recombinant, stereospecific monoterpene synthases, a (-)-limonene synthase (CsTPS1) and a (+)-α-pinene synthase (CsTPS2), encoded by Cannabis sativa L. cv. ‘Skunk’ trichome mRNA, have been isolated and characterized. Recombinant CsTPS1 showed a Kmvalue of 6.8 μM, a Vmaxof 1.1 × 10−4μmol/min and Vmax/Kmof 0.016; the pH optimum was determined at pH 6.5, and a temperature optimum at 40°C. Recombinant CsTPS2 showed a Kmvalue of 10.5 μM, a Vmaxof 2.2 × 10−4μmol/min and Vmax/Kmof 0.021; the pH optimum was determined at pH 7.0, and a temperature optimum at 30°C. Phylogenetic analysis showed that both CsTPSs group within the angiosperms and belong to the Tpsb subgroup of monoterpene synthases. The enzymatic products (-)-limonene and (+)-α-pinene were detected as natural products in C. sativa trichomes.

Zoledronic acid (ZOL), an aminobisphosphonate, is used in the clinic to prevent pathologic resorption of bone by osteoclasts in multiple myeloma. ZOL may also have direct anti-myeloma activity, perhaps through inhibition of farnesyl pyrophosphate (FPP) synthase. FPP synthase is an enzyme in the isoprenoid biosynthetic pathway (step 1 in figure). This pathway is responsible for the production of isoprenoid pyrophosphates. These may be covalently linked to small GTPases; the addition of the 15 carbon farnesyl moiety is termed farnesylation (step 2) and the 20 carbon geranylgeranyl moiety is geranylgeranylation (step 3). These post-translational modifications of small GTPases allow for proper membrane association that is necessary for normal signal transduction. To further elucidate the role of FPP synthase inhibition as a potential mechanism of action of ZOL, a human derived myeloma cell line, RPMI-8226 was incubated with 50 microM ZOL for 24 hours. ZOL diminished isoprenylation of Ras, Rab 6 and Rap 1A, as determined by Western blotting. Addition of 10 microM of geranylgeranyl pyrophosphate (GGPP) restored processing of these small GTPases. Processing was not restored with the addition of 1mM of mevalonate, 10 microM geranyl pyrophosphate (GPP), or 10 microM farnesyl pyrophosphate. As these latter components are upstream of FPP synthase these data suggest that ZOL may act by limiting availability of GGPP for isoprenylation. This is further supported by treatment of RPMI-8226 cells with a novel GGPP synthase inhibitor (step 4), which can be reversed by addition of GGPP but not by FPP. To more accurately mimic the marrow microinvironment, RPMI-8226 cells were incubated in an in vitro co-culture with murine derived osteoclasts. Briefly, bone marrow was harvested from C57B1/6 mice by flushing the long bones with culture media using a 31g needle. The cell suspension was centrifuged for 10 minutes and the cell pellet was resuspended in alpha-MEM containing 10% fetal bovine serum, 20 nM murine GM-CSF, 25nM PTHrP, 50 nM ascorbic acid, 10 nM Vitamin D3 and 1% penicillin/streptomycin. The bone marrow from two long bones was seeded in each 3.5 cm well and non-adherent cells were removed after 24 hours. This created a feeder layer which included osteoclasts, confirmed by TRAP staining. RPMI-8226 (4x106 cells/well) were added to the feeder layer on day six of the culture. The RPMI-8226 cells, normally a suspension cell line, became adherent to the feeder layer by 24 hours. They exhibited >95% viability confirmed by trypan blue exclusion. Under these conditions ZOL impaired protein isoprenylation by reducing geranylgeranylation but not farnesylation. These data further support the critical role of geranylgeranylation but not farnesylation of some small GTPases in myeloma cells. The co-culture system allows for further evaluation of the requirements of protein isoprenylation in myeloma cells and serves as an in vitro model to evaluate the impact of manipulating the isoprenoid pathway on osteoclast-myeloma interactions. Figure Figure

Thermal and Enzymatic Conversions of Precursors to Flavor Compounds: An Overview Monoterpene Biosynthesis: The Cyclization of Geranyl Pyrophosphate to (+)-Sabinene Lipases: Useful Biocatalysts for Enantioselective Reactions of Chiral Flavor Compounds Carboxylester-Lipase Mediated Reactions: A Versatile Route to Target the Chiral Molecules Biosynthesis and Studies on the Biotechnological Production of Aliphatic *g- and *d-Lactones Precursor Atmospheric Technology: Efficient Aroma Enrichment in Fruit Cells Glycosidic Precursors of Varietal Grape and Wine Flavor Glucosides of Limonoids Oxytenated C[1[3-Norisoprenoids: Important Flavor Precursors Free and Bound Flavor Constituents of White-Fleshed Nectarine Thermally Degraded Thiamin: A Potent Source of Interesting Flavor Compounds Studies on the Formation of Furaneol in Heat-Processed Foods Flavor Compounds Formed from Lipids by Heat Treatment Formation of Meat-like Flavor Compounds Peptides as Flavor Precursors in Model Maillard Reactions Meat Flavor Generation from Cysteine and Sugars Analysis and Reactivity of Glucosones Formation of Smoke Flavor Compounds by Thermal Lignin Degradation Reaction Kinetics for the Formation of Heterocyclic Compounds in a Temperature and Pressure Controlled Experiment

Farnesyl pyrophosphate (FPP) synthetase catalyzed the condensation of geranyl pyrophosphate (GPP) with 4-methyl-4-pentenyl pyro phosphate in the presence of Ni2+ to give an unexpected product, homofarnesyl pyrophosphate having an exomethylene group, together with Z-homofarnesyl pyrophosphate.

To determine whether the bicyclic monoterpene olefins (-)-alpha-pinene and (-)-beta-pinene arise biosynthetically from the same monoterpene cyclase by alternate deprotonations of a common carbocationic intermediate, the product distributions arising from the acyclic precursor (10-/sup 2/H/sub 3/,1-/sup 3/H)geranyl pyrophosphate were compared with those resulting from incubation of (1-3H)geranyl pyrophosphate with (-)-pinene cyclase from Salvia officinalis. Alteration in proportions of the olefinic products generated by the partially purified pinene cyclase resulted from the suppression of the formation of (-)-beta-pinene (C10 deprotonation) by a primary deuterium isotope effect with a compensating stimulation of the formation of (-)-alpha-pinene (C4 deprotonation). (-)-Pinene cyclase as well as (+)-pinene cyclase also exhibited a decrease in the proportion of the acyclic olefin myrcene generated from the deuteriated substrate, accompanied by a corresponding increase in the commitment to cyclized products. The observation of isotopically sensitive branching, in conjunction with quantitation of the magnitude of the secondary deuterium isotope effect on the overall rate of product formation by the (+)- and (-)-pinene cyclases as well as two other monoterpene cyclases from the same tissue, supports the biosynthetic origin of (-)-alpha-pinene and (-)-beta-pinene by alternative deprotonations of a common enzymatic intermediate. A biogenetic scheme consistent with these results is presented, andmore » alternate proposals for the origin of the pinenes are addressed.« less

Conversion of geranyl pyrophosphate to cyclic monoterpenes is considered to involve the preliminary isomerization of this acyclic precursor to enzyme-bound linalyl pyrophosphate and the cyclization of this tertiary intermediate. 2-Fluorogeranyl pyrophosphate and 2-fluorolinalyl pyrophosphate are effective competitive inhibitors of the cyclization of geranyl pyrophosphate by several different monoterpene cyclases, and the electron withdrawing α-fluorine substituent was shown to suppress the rate of cyclic product formation from both tritium-labeled analogs by at least two orders of magnitude. These results indicate that both steps of the coupled isomerization-cyclization sequence are initiated by ionization of an allylic pyrophosphate, and they confirm the electrophilic nature of this enzymatic reaction type and its similarity to the prenyltransferase reaction.

Isopentenyl pyrophosphate isomerase, farnesyl pyrophosphate synthetase, and geranylgeranyl pyrophosphate synthetase were detected in cell-free extracts of Bombyx mori and were partially purified by hydroxyapatite and Sephadex G-100 chromatography. Two forms of farnesyl pyrophosphate synthetase were chromatographically separated. They were designated as farnesyl pyrophosphate synthetases I and II in the order of their elution from hydroxyapatite. Both enzymes catalyzed the exclusive formation of (E,E)-farnesyl pyrophosphate from isopentenyl pyrophosphate and either dimethylallyl pyrophosphate or geranyl pyrophosphate. However, they were not interconvertible, unlike the enzyme from pig liver. These two enzymes resembled each other in pH optima and molecular weights but differed in susceptibility to metal ions. Farnesyl pyrophosphate synthetase II was stimulated by Triton X-100 while synthetase I was inhibited by the same reagent.

The effect of the fungicide diniconazole on the growth and volatile terpene content of garden sage and scotch spearmint was tested. The growth of both plant species was stunted, and the degree of stunting was dose dependent. Monoterpene levels on a per leaf basis were increased at all dosages in the case of sage, whereas in scotch spearmint lower dosages increased monoterpene levels, but higher dosages or repeated applications reduced them. Monoterpene composition was also altered by diniconazole treatment of sage. The results suggest that monoterpene biosynthesis was influenced either at the cyclization step of monoterpene formation and/or at a stage prior to the committed cyclization of geranyl pyrophosphate. The cytochrome P450-dependent hydroxylation of cyclic monoterpene olefins was not influenced by diniconazole.

(+)-Pinene cyclase from sage (Salvia officinalis) catalyzes the isomerization and cyclization of geranyl pyrophosphate to (+)-alpha-pinene and (+)-camphene, and to lesser amounts of (+)-limonene, myrcene, and terpinolene, whereas (-)-pinene cyclase from this tissue catalyzes the conversion of the acyclic precursor to (-)-alpha-pinene, (-)-beta-pinene, and (-)-camphene, and to lesser quantities of (-)-limonene, myrcene, and terpinolene. The bicyclic products of these enzymes (pinene and camphene) are derived via the cyclization of the cisoid, anti-endo-conformers of the bound, tertiary allylic intermediates (3R)-linalyl pyrophosphate [+)-pinene cyclase) and (3S)-linalyl pyrophosphate [-)-pinene cyclase). When challenged with either enantiomer of linalyl pyrophosphate or with neryl pyrophosphate (cis-isomer of geranyl pyrophosphate) as substrate, both pinene cyclases synthesize disproportionately high levels of acyclic olefins (myrcene and ocimene) and monocyclic olefins (limonene and terpinolene), compared with the product mixtures generated from the natural geranyl precursor. Resolution of the limonene derived from linalyl pyrophosphate and neryl pyrophosphate demonstrated that this monocyclic olefin was formed via conformational foldings in addition to the cisoid,anti-endo-pattern. These results indicate that the alternate substrates are ionized by the cyclases prior to their achieving the optimum orientation for bicyclization. In the case of geranyl pyrophosphate, a preassociation mechanism is suggested in which optimum folding of the terpenyl chain precedes the initial ionization step.

It has been shown that a 10 000 x g matrix-free mitochondrial membrane-rich preparation from commercial bakers' yeast is able to synthesize 3-all-transhexaprenyl-4-hydroxybenzoate from 4-hydroxybenzoate and isopentenyl pyrophosphate. The synthesis is Mg2+ dependent and is stimulated markedly by the primer for polyprenylpyrophosphate synthesis of 3-hexaprenyl-4-hydroxybenzoate from 4-hydroxybenzoate, isopentenyl pyrophosphate and 3,3-dimethylallyl pyrophosphate the priming function of 3,3-dimethylallyl pyrophosphate can be performed by either geranyl pyrophosphate (most efficient) or farnesyl pyrophosphate. At high Mg2+ concentrations, however, geranyl pyrophosphate and farnesyl pyrophosphate act mainly as sources of preformed side chains and 3-diprenyl- and 3-tripenyl-4-hydroxybenzoate, respectively, are produced. In the presence of a source of preformed polyprenyl pyrophosphates the membrane preparations catalysed the polyprenylation of methyl-4-hydroxybenzoate, 4-hydroxybenzaldehyde, 4-hydroxybenzylalcohol and 4-hydroxycinnamate. No evidence was obtained for the involvement of either 4-hydroxybenzoyl CoA or 4-hydroxybenzoyl-S-protein in the formation of 3-polyprenyl-4-hydroxybenzoates.

A partially purified and lyophilized preparation of (+)-alpha-pinene cyclase from sage (Salvia officinalis) was shown to convert geranyl pyrophosphate to the monoterpene olefins alpha-pinene, camphene, limonene, and myrcene, in hexane with the addition of 0.1 to 10% water. This constitutes the first report of catalysis by a terpene cyclase in "non-aqueous" media. The relative proportions of olefinic products, metal ion requirement, pH optimum, temperature response, and time course of the enzymatic conversion were determined. The cyclase was shown to be stabilized with respect to temperature and time by the use of hydrocarbon solvent, and, in all other characteristics, to exhibit properties closely resembling those observed in aqueous media.

A prenyltransferase activity (EC 2.5.1.1) has been partially purified from the flavedo of Citrus sinensis with 30–40-fold purification and 35–60 % yield. The enzyme catalyses the condensation of IPP with DMAPP or GPP. The products are neryl and geranyl pyrophosphate as well as (2 E ,6 E )- and (2 Z ,6 E )-farnesyl pyrophosphate. The two C 15 -products are predominant. The E - and Z -synthetase activities are partially dissociated during the purification procedure, as well as by heat or ageing. Preparations devoid of Z -synthetase were obtained. Mg 2 + is required for full activity. Mn 2 + or Co 2 + can replace Mg 2 + . The ratio of E/Z -products formed is different for each cation. Mg 2 + complexes of allylic substrates or of products protect the enzyme against heat-inactivation and against inactivation by DTNB. The results are interpreted in terms of two or more prenyltransferases stereoselective for the synthesis of E - and Z -products.

Phosphoric esters bearing a sulfonium group in the γ position are converted under basic conditions into allylic phosphates.

The mechanism of the 1'-4 coupling reaction between isopentenyl pyrophosphate and geranyl pyrophosphate catalyzed by farnesyl pyrophosphate synthetase from porcine liver was studied with the allylic substrate analogue 2-fluorogeranyl pyrophosphate. 2-Fluorogeranyl pyrophosphate is an alternate substrate for the enzyme, yielding 6-fluorofarnesyl pyrophosphate upon condensation with isopentenyl pyrophosphate. The Michaelis constant for the fluoroanalogue, Km = 1.1 micron, is similar to that measured for geranyl pyrophosphate, Km = 0.7 micron. However, the rate of condensation with the fluoroanalogue was only 8.4 X 10(-4) that of the normal reaction. A similar rate of depression (4.4 X 10(-3)) was found for solvolysis of geranyl methanesulfonate and the corresponding 2-fluoro derivative, reactions known to proceed via cationic intermediates. In contrast, displacement of chlorine from geranyl chloride and 2-fluorogeranyl chloride by cyanide showed a small (2-fold) rate enhancement for the fluoro compound. Finally, 2-fluorogeranyl pyrophosphate is a competitive inhibitor against geranyl pyrophosphate. These data are interpreted in terms of an ionization-condensation-elimination mechanism for the 1'-4 coupling reaction.

The primary monoterpene accumulated in the glandular trichomes of spearmint (Mentha spicata) is the ketone (-)-carvone which is formed by cyclization of the C(10) isoprenoid intermediate geranyl pyrophosphate to the olefin (-)-limonene, hydroxylation to (-)-trans-carveol and subsequent dehydrogenation. Selective extraction of the contents of the glandular trichomes indicated that essentially all of the cyclase and hydroxylase activities resided in these structures, whereas only about 30% of the carveol dehydrogenase was located here with the remainder located in the rest of the leaf. This distribution of carveol dehydrogenase activity was confirmed by histochemical methods. Electrophoretic analysis of the partially purified carveol dehydrogenase from extracts of both the glands and the leaves following gland removal indicated the presence of a unique carveol dehydrogenase species in the glandular trichomes, suggesting that the other dehydrogenase found throughout the leaf probably utilizes carveol only as an adventitious substrate. These results demonstrate that carvone biosynthesis takes place exclusively in the glandular trichomes in which this natural product accumulates.

(1R)-1-3H-labeled and (1S)-1-3H-labeled geranyl pyrophosphate and neryl pyrophosphate were prepared from the corresponding 1-3H-labeled aldehydes by a combination of enzymatic and synthetic procedures. Following admixture with the corresponding 2-14C-labeled internal standard, each substrate was converted to (+)-bornyl pyrophosphate and (-)-bornyl pyrophosphate by cell-free enzyme preparations from sage (Salvia officinalis) and tansy (Tanacetum vulgare), respectively. Each pyrophosphate ester was hydrolyzed, and the resulting borneol was oxidized to camphor. The stereochemistry of labeling at C-3 of the derived ketone was determined by base-catalyzed exchange, taking advantage of the known selective exchange of the exo-alpha-protons. By comparison of such exchange rates to those of product generated from (1RS)-2-14C,1-3H2-labeled substrate, it was demonstrated that geranyl pyrophosphate was cyclized to bornyl pyrophosphate with net retention of configuration at C-1 of the acyclic precursor, whereas neryl pyrophosphate was cyclized to product with inversion of configuration at C-1. The observed stereochemistry is consistent with a reaction mechanism whereby geranyl pyrophosphate is first stereospecifically isomerized to linalyl pyrophosphate which, following rotation about C-2-C-3 to the cisoid conformer, cyclizes from the anti-endo configuration. Neryl pyrophosphate cyclizes either directly or via the linalyl intermediate without the attendant rotation.

Prenyltransferase (dimethylallydiphosphate: isopentenyldiphosphate dimethylallytransferase, EC 2.5.1.1) has been purified to homogeneity from human liver obtained at autopsy. The enzyme is a dimer with a native molecular weight of 74 000 +/- 1 400. The amino acid composition is reported. the enzyme has a broad pH optimum between 7.3 and 8.8 and an absolute requirement for either Mn2+ or Mg2+ for activity; half-maximal activity was observed at 3.7 microM Mn2+ or 89.0 microM Mg2+. Michaelis constants for geranyl pyrophosphate and isopentenyl pyrophosphate were 0.44 and 0.94 microM, respectively; the V value for synthesis of farnesyl pyrophosphate from these substrates was 1.1 mumol . min-1 . mg-1. Isopentenyl pyrophosphate inhibited the reaction rates at concentrations above 2 microM when the concentrations of geranyl pyrophosphate were less than 2 microM. The highest concentration of geranyl pyrophosphate tested, 16 microM, showed no inhibition of reaction rates even when the concentration of isopentenyl pyrophosphate was as low as 0.2 microM. Only one form of human liver prenyltransferase could be observed under conditions which resolved the porcine enzyme into two distinct forms; the human enzyme is akin, physico-chemically, to the B-form of the pig liver enzyme. After dialysis against Tris-HCl buffer, pH 7.8, the enzyme became completely dependent upon dithiols or thiols for its activity. Kinetic experiments with a partially activated enzyme sample showed that the activation by the dithiol greatly enhanced the affinity of the enzyme for geranyl pyrophosphate, but not that for isopentenyl pyrophosphate. The human prenyltransferase is inactivated by phenylglyoxal according to pseudo-first-order kinetics, but is protected against the inactivation by 3,3-dimethylallyl and geranyl pyrophosphate. It is also inactivated by high concentrations (greater than 2 mM) of iodoacetic acid, but is protected against the inactivation by dithiothreitol. Antibodies raised to the B-form of the pig liver enzyme cross-reacted with the human prenyltransferase and were 47% as effective in precipitating the human enzyme as the porcine enzyme. In double immunodiffusion experiments the antiserum was monospecific against the B-form of the porcine enzyme; it also gave a single precipitin line with the A-form, but not identical with that given by the B-form. It gave a precipitin line also with the human enzyme, but not identical with that given by either the A- or B-form of the porcine enzyme.

The volatile oil of sage ( Salvia officinalis ) leaf contains primarily cyclic monoterpenes, and a cell-free enzyme system prepared from immature sage leaves catalyzed the conversion of [1- 3 H]neryl pyrophosphate to cyclic monoterpenes, including 1,8-cineole, α-terpineol, and limonene. The identity of these products was confirmed by the preparation of derivatives, chemical degradation studies, and radiochromatographic analyses. Enzymatic activity capable of 1,8-cineole and hydrocarbon formation was located primarily in the soluble fraction of the leaf homogenate, whereas activity responsible for α-terpineol formation, and phosphatase activity, was distributed among the soluble fraction and the 3000g particles. The formation of all cyclic products was stimulated in the presence of MnCl 2 (1–2 m m ) and MgCl 2 (5–10 m m ) and by inclusion of phosphatase inhibitors, such as NaF, in the incubation medium. Enzymatic activity increased linearly with protein concentration and time and was maximum at about pH 6.5. As α-terpineol could give rise to limonene by dehydration and yield 1,8-cineole by intramolecular attack of the hydroxyl at the double bond, the possible intermediary role of α-terpineol was examined. Inclusion of 1 m m (±)-α-terpineol in the incubation medium suppressed 1,8-cineole and hydrocarbon formation from neryl pyrophosphate by approximately 35%, suggesting that this alcohol could be an intermediate. However, the inhibition appeared to be nonspecific as several other monoterpene alcohols were similarly effective. In order to provide a more direct examination of the pathway, (±)-[3- 3 H]α-terpineol was prepared and tested as a substrate with the enzyme system. (±)-[3- 3 H]α-Terpineol was not converted to 1,8-cineole or to cyclic hydrocarbons, indicating that this alcohol was not an intermediate in the biosynthesis of the structurally related compounds. Furthermore, labeled α-terpineol was not incorporated into other monoterpenes in sage leaf slices, whereas the acyclic alcohols, [1- 3 H]nerol and [1- 3 H]geraniol, were readily incorporated into the characteristic cyclic monoterpenes of sage leaf. Similar lines of evidence excluded 1,8-cineole, limonene, and terpinolene as intermediates in the formation of cyclic products from neryl pyrophosphate. Thus, all of these results suggest that 1,8-cineole, α-terpineol, and limonene are derived independently from neryl pyrophosphate, rather than as free intermediates of a common reaction sequence. Additionally, this is the first report on the biosynthesis of 1,8-cineole in a cell-free system.