Substrate and metal specificity in the enzymic synthesis of cyclic monoterpenes from geranyl and neryl pyrophosphate

A partially purified enzyme (carbocyclase) from the flavedo of Citrus limonum formed α-pinene, β-pinene, limonene, and γ-terpinene from geranyl pyrophosphate (GPP) and neryl pyrophosphate. The maximum specific activities obtained were 7.0 and 3.6 nmol/ min/mg, respectively. Cross-inhibition by the two substrates were observed and the ability to utilize neryl pyrophosphate was almost completely lost with aging. Citronellyl pyrophosphate and dimethylallyl pyrophosphate were the most effective inhibitors of carbocyclase. Isopentenyl pyrophosphate, the monophosphate esters of nerol and geraniol, as well as inorganic pyrophosphate were much less effective inhibitors. The enzyme had an absolute requirement for Mn²⁺. It could be replaced with about 2% effectiveness by Mg²⁺ and Co²⁺. Kinetic studies showed that the observed reaction rate correlates with the calculated concentration of the GPP (Mn²⁺)₂ species. Previous evidence with nonenzymatic reactions and the results presented support the view that the mechanism of carbocyclase may be the intramolecular analog of prenyltransferase.     

Carotene biosynthesis by a cell extract of Aspergillus giganteus mut alba

A cell extract prepared from lyophilized mycelia of light-grown cultures of Aspergillus giganteus mut alba converted [2-¹⁴C]mevalonic acid into phytoene, lycopene, β-carotene and squalene, but from similar preparations from dark grown cultures formed only squalene. The carotenogenic activities of the cell extracts varied with the age of the cultures. Phytoene synthetase was located in the cytosolic fraction, whereas the dehydrogenation and cyclisation steps were catalysed by membrane-bound enzymes. Dithiothreitol, ATP, Mn²⁺, Mg²⁺, NAD and NADP were essential for the formation of carotenes from mevalonic acid, whilst FAD was required for phytoene metabolism. Oxygen enhanced the conversion of phytoene into other carotenes.     

Biosynthesis of ent-kaurene in cell-free extracts of Pisum sativum shoot tips

Soluble enzyme preparations from pea shoot tips incorporated mevalonic acid-2-¹⁴C into ent-kaurene-¹⁴C, squalene-¹⁴C and other products. The assay for either ent-kaurene or squalene is quite direct; both products can be obtained apparently free of radioactive contaminants by TLC on silica gel G in hexane. The enzyme system is dependent upon added ATP and Mn²⁺ or Mg²⁺, with Mn²⁺ being a more effective activator than Mg²⁺ under the experimental conditions. Reduced pyridine nucleotide had no effect on ent-kaurene production but stimulated squalene synthesis. The accumulation of both ent-kaurene and squalene was stimulated by dithiothreitol and carbon monoxide and was reduced by the addition of particulate cell components. AMO-1618 inhibited ent-kaurene production and had no effect on the synthesis of squalene. Enzyme extracts from shoot tips are much less active in ent-kaurene synthesis than extracts from the cotyledons of immature seeds on either a fresh weight or protein basis.     

Lactobacillus plantarum undecaprenyl pyrophosphate synthetase: purification and reaction requirements.

Undecaprenyl pyrophosphate synthetase was partially purified from Lactobacillus plantarum by DEAE-cellulose, hydroxyapatite, and Sephadex G-100 chromatography in Triton X-100. The enzyme has a molecular weight between 53,000 and 60,000. The enzyme demonstrated a fivefold preference for farnesyl pyrophosphate rather than geranyl pyrophosphate as the allylic cosubstrate, whereas dimethylallyl pyrophosphate was not effective as a substrate. Polyprenyl pyrophosphates obtained using either farnesyl or geranyl pyrophosphate as cosubstrate were chromatographically identical. Hydrolysis of these polyprenyl pyrophosphates with either a yeast or liver phosphatase preparation yielded undecaprenol as the major product. Incorporation of radioactive label from mixtures of Δ³-[1-¹⁴C]isopentenyl pyrophosphate and Δ³-2R-[2-³H]isopentenyl pyrophosphate into enzymic product indicated that each isoprene unit added to the allylic pyrophosphate substrate has a cis configuration about the newly formed double bond. The removal of detergent from enzyme solutions resulted in a parallel loss in enzyme activity when analyzed with either farnesyl or geranyl pyrophosphate as cosubstrates. Enzymic activity was restored on addition of Triton X-100 or deoxycholate. The enzyme exhibited a pH-activity profile with optima at pH 7.5 and 10.2. It also demonstrated a divalent cation requirement, with Mg²⁺, Mn²⁺, Zn²⁺, and Co²⁺ exhibiting comparable activities.     

Squalene biosynthesis by a cell-free extract of Rhizopus arrhizus

Squalene was identified as the principal radioactive component (85%) of the hexane-soluble products formed from mevalonate-[2-¹⁴C] by a cell-free preparation from Rhizopus arrhizus. The system required ATP, NADH and Mn²⁺ to obtain 20–40% incorporation of the substrate into squalene. Temperature and pH optima for the system were 20° and 7.0, respectively.     

Monoterpene hydrocarbon biosynthesis by isolated leucoplasts of Citrofortunella mitis

A plastid vesicle preparation isolated from exocarpium of young Citrofortunella mitis (calamondin) fruits was able to synthesise monoterpene hydrocarbons when incubated with isopentenyl pyrophosphate. The electron-microscope comparison between this organelle fraction and the various plastid classes present in the peel tissues has shown the structural identity between these plastid vesicles and the leucoplasts of the epithelial cells lining the secretory pockets. The monoterpene biosynthesis required the presence of dimethylallyl pyrophosphate, Mn²⁺ or Mg²⁺ and was increased by addition of 2-mercaptoethanol. Evidence is provided that the leucoplast vesicles act as a complete system in which occur all the successive steps involved in monoterpene hydrocarbon elaboration from isopentenyl pyrophosphate.     

Isopentenyl pyrophosphate isomerase and prenyltransferase from tomato fruit plastids

Isopentenyl pyrophosphate isomerase has been isolated from an extract of tomato fruit plastids and purified 245-fold by fractionation with ammonium sulfate, gel filtration on Bio-Gel A 1.5m, ion-exchange chromatography on DEAE-cellulose, gel filtration on Sephadex G-100, and chromatofocusing. Gel filtration on Sephadex G-100 separated the isopentenyl pyrophosphate isomerase from a prenyltransferase fraction that catalyzed the conversion of isopentenyl pyrophosphate to acid-labile compounds in the presence of dimethylallyl, geranyl, or farnesyl pyrophosphates. The molecular weights of the isopentenyl pyrophosphate isomerase and prenyltransferase were determined to be 34,000 and 64,000, respectively, by gel filtration on Sephadex G-100. The only cofactor required by either the isomerase or the prenyltransferase was a divalent cation, either Mg²⁺ or Mn²⁺. Isopentenyl pyrophosphate isomerase could also be totally inactivated by 1 × 10^?3m iodoacetamide, and this property was utilized in the assay of prenyltransferase activity in the presence of contaminating isomerase. The inactivation of isomerase by iodoacetamide is consistent with the stabilization of isopentenyl pyrophosphate isomerase by dithiothreitol. The K_m of isopentenyl pyrophosphate isomerase for isopentenyl pyrophosphate was found to be 5.7 × 10^?6.     

Synthesis of monoterpene hydrocarbons from [1-3H]linalyl pyrophosphate by carbocyclase from Citrus limonum

A partially purified enzyme preparation from the flavedo of Citrus limonum utilized [1-³H]linalyl pyrophosphate as a substrate for cyclic terpene hydrocarbon formation more efficiently than the pyrophosphates of nerol and geraniol. The products formed from all three substrates are α-pinene, β-pinene, limonene, and γ-terpinene. Neryl and geranyl pyrophosphate inhibit the formation of these products from linalyl pyrophosphate. No free linalyl pyrophosphate could be detected during the enzymatic formation of cyclic terpene hydrocarbons from geranyl pyrophosphate. Mn²⁺ catalyzes the nonenzymatic solvolysis of linalyl pyrophosphate, forming myrcene and ocymenes and no bicyclic hydrocarbons. Linalyl pyrophosphate is a sterically plausible precursor of cyclic hydrocarbons, but the present data support only its role as an alternative substrate and not as an obligatory free intermediate in terpene biosynthesis.     

Isopentenyl pyrophosphate isomerase from pig liver

Isopentenyl pyrophosphate isomerase (EC 5.3.3.2) from pig liver has been purified 197-fold. The preparation was estimated to contain less than 10% of contaminating protein. The molecular weight determined by gel filtration was 82,500 ± 3,000 and the isoelectric point from isoelectric focusing was in the range 6.0–6.2. N-terminal analysis showed the presence of both leucine and proline. The pH optimum of the enzyme preparation was 6.3. After dialysis against EDTA, activity was restored by either Mn²⁺ or Mg²⁺, the former being more effective. At the optimum pH and concentration of Mn²⁺, K_m and V were 2.7 μm and 6.7 μmol min^?1 mg^?1, respectively. The enzyme was partially inhibited by a variety of terpene mono- and pyrophosphate esters, by inorganic phosphate ions, and by acetate ions; essentially complete inhibition by sulfhydryl-blocking reagents was observed. ATP partially inhibited, the degree of inhibition showing a sigmoid dependence on ATP concentration. Monothiols and dithiothreitol activated the enzyme, as did mevalonic acid.     

Biosynthesis of monoterpene hydrocarbons from [1-3H]neryl pyrophosphate and [1-3H]geranyl pyrophosphate by soluble enzymes from Citrus limonum.

A soluble enzyme preparation from the flavedo of Citrus limonum transforms [1-³H₁]neryl pyrophosphate or [1-³H₁]geranyl pyrophosphate into β-pinene, sabinene, α-pinene, and limonene. The enzyme has been partially purified and stabilized by precipitation with polyethyleneglycol. The enzymic cyclization requires the presence of Mn²⁺, which cannot be replaced with Mg²⁺. The addition of reagents containing sulfhydryl groups is essential for optimal activity. Allylic C₁₀ monophosphates do not act as substrates, but they inhibit hydrocarbon formation. Inorganic pyrophosphate has a similar inhibitory effect. No interconversion of neryl and geranyl pyrophosphate has been observed. Possible pathways for the enzymic cyclization reactions are proposed.     

Biosynthesis of squalene and other triterpenes in Mentha piperita from mevalonate-2-14C

Unlike mono- and sesqui-terpenes, squalene and other triterpenes in peppermint readily incorporate mevalonate-2-¹⁴C label (greater than 30% incorporation of R-mevalonate in 4 hr). The labelled squalene produced turns over rapidly. Squalene derived from mevalonate-2-¹⁴C in incorporation times of 1, 4 and 7 hr was degraded chemically and shown to be equivalently labelled, according to theory, in the isopentenyl pyrophosphate-derived and dimethylallyl pyrophosphate-derived portions of the molecule. This contrasts with earlier studies on the biosynthesis of mono- and sesqui-terpenes in peppermint from ¹⁴C-precursors, in which the isopentenyl pyrophosphate-derived portions of the terpene molecules were found to be preferentially labelled, suggesting the presence of endogenous dimethylallyl pyrophosphate pools. The kinetics of squalene biosynthesis, and the labelling pattern of squalene, suggest that sites of triterpene biosynthesis are readily accessible to exogenous mevalonate and that endogenous dimethylallyl pyrophosphate pools do not participate in triterpene biosynthesis to any appreciable extent. The triterpene biosynthetic sites in peppermint thus appear to differ significantly from the monoterpene and sesquiterpene biosynthetic sites.     

Purification and characterization of pyruvate dehydrogenase complex from borccoli floral buds.

The pyruvate dehydrogenase complex (PDC) was purified from Brassica oleracea var. italica floral buds to a specific activity of approximately 6 μmol of NADH formed/min/ mg of protein. The PDC had cofactor requirements for NAD⁺, thiamine pyrophosphate, coenzyme A, and a divalent cation (Mg²⁺, Ca²⁺, or Mn²⁺). The enzyme catalyzed the oxidative decarboxylation of pyruvate at a rate threefold faster than 2-oxobutyrate but was inactive toward 2-oxoglutarate. The PDC was competively inhibited by acetyl-CoA against CoA and NADH against NAD⁺. The enzyme was shown to be more sensitive to regulation by NADH than acetyl-CoA.     

Biosynthesis of monoterpenes: Preliminary characterization of l-endo-fenchol synthetase from fennel (Foeniculum vulgare) and evidence that no free intermediate is involved in the cyclization of geranyl pyrophosphate to the rearranged product

A soluble enzyme preparation from the leaves of fennel (Foeniculum vulgare M.) has been shown to catalyze the cation-dependent cyclization of both geranyl pyrophosphate and neryl pyrophosphate to the bicyclic rearranged monoterpene l-endo-fenchol (R. Croteau, M. Felton, and R. Ronald, 1980 Arch. Biochem. Biophys.200, 524–533). To examine the possible presence of free intermediates between the acyclic precursors and fenchol, and to remove competing cyclase and pyrophosphatase activities, the soluble preparation was partially purified by ammonium sulfate fractionation followed by gel filtration on Sephadex G-150 and ion exchange chromatography on O-diethylaminoethyl-cellulose. Activities for the cyclization of geranyl pyrophosphate and neryl pyrophosphate to fenchol were coincident on Chromatographic fractionation suggesting that the same enzyme was capable of cyclizing both acyclic substrates. No interconversion of the acyclic precursors was detected. Although bornyl pyrophosphate is a free intermediate in the biosynthesis of the related bicyclic monoterpenol borneol, both protein fractionation and isotopic dilution experiments ruled out endo-fenchyl pyrophosphate as a free intermediate in fenchol biosynthesis. Similarly, while construction of the fenchane skeleton was demonstrated to involve the rearrangement of an intermediate pinane skeleton, isotopic dilution experiments ruled out both optical antipodes of α-pinene, β-pinene, cis-2-pinanol, trans-2-pinanol, and the corresponding 2-pinyl pyrophosphates as free intermediates of the enzyme-catalyzed reaction. Furthermore, exhaustive search of the enzymatic reaction products provided no evidence to suggest the involvement of any free intermediate between the acyclic precursor and fenchol. The endo-fenchol synthetase has an apparent molecular weight of 60,000, shows a pH optimum near 7.0, and requires Mn²⁺ (1 mm) for catalytic activity. Co²⁺ can partially substitute for Mn²⁺, but other divalent cations are ineffective. The partially purified synthetase is inhibited by p-hydroxymercuribenzoate and by phenylglyoxal, and it exhibits a preference for geranyl pyrophosphate over neryl pyrophosphate as substrate. An integrated scheme is proposed for the cyclization and rearrangement catalyzed by fenchol synthetase.     

Construction of carotenoid biosynthetic pathways using squalene synthase

The first committed steps of steroid/hopanoid pathways involve squalene synthase (SQS). Here, we report the Escherichia coli production of diaponeurosporene and diapolycopene, yellow C₃₀ carotenoid pigments, by expressing human SQS and Staphylococcus aureus dehydrosqualene (C₃₀ carotenoid) desaturase (CrtN). We suggest that the carotenoid pigments are synthesized mainly via the desaturation of squalene rather than the direct synthesis of dehydrosqualene through the non-reductive condensation of prenyl diphosphate precursors, indicating the possible existence of a “squalene route” and a “lycopersene route” for C₃₀ and C₄₀ carotenoids, respectively. Additionally, this finding yields a new method of colorimetric screening for the cellular activity of squalene synthases, which are major targets for cholesterol-lowering drugs.     

The partial purification and properties of a phytoene synthesizing enzyme system.

An enzyme system catalyzing the synthesis of phytoene from isopentenyl pyrophosphate has been isolated from tomato fruit plastids and purified approximately 350-fold in specific activity. This enzyme system has a molecular weight of approximately 200,000. The rate of phytoene formation is maximal at pH 7.0 and 23 °C and the apparent K_m for isopentenyl pyrophosphate is 10 μm The rates of phytoene synthesis when geranylgeranyl pyrophosphate and isopentenyl pyrophosphate were used as substrates were 0.08 and 0.17 nmol of phytoene/mg of protein/h, respectively. The enzyme complex showed an absolute requirement for Mn²⁺, but not for NADP⁺. At a concentration of 2 mm, NADP⁺ produced only a 1.5- to 3-fold stimulation, and this effect varied from preparation to preparation. The addition of NADPH to the incubation mixture produced inhibition of phytoene synthesis and there was no evidence for the concomitant accumulation of lycopersene. The acid labiles produced on acid treatment of the incubation mixture indicated that geranylgeranyl pyrophosphate was formed by the enzyme complex. The enzyme system is stabilized in the presence of 30% glycerol and 10 mm dithiothreitol and it can be stored at ?20 °C for over 1 month without significant loss of activity. However, the enzyme activity for phytoene formation is heat labile, and it is not stable when attempts are made to purify it further by ion-exchange chromatography.     

Biosynthesis of monoterpenes: preliminary characterization of bornyl pyrophosphate synthetase from sage (Salvia officinalis) and demonstration that Geranyl pyrophosphate is the preferred substrate for cyclization.

Previous studies with soluble enzyme preparations from sage (Salvia officinalis) demonstrated that the monoterpene ketone (+)-camphor was synthesized by the cyclization of neryl pyrophosphate to (+)-bornyl pyrophosphate followed by hydrolysis of this unusual intermediate to (+)-borneol and then oxidation of the alcohol to camphor (R. Croteau, and F. Karp, 1977, Arch. Biochem. Biophys.184, 77–86). Preliminary investigation of the (+)-bornyl pyrophosphate synthetase in crude preparations indicated that both neryl pyrophosphate and geranyl pyrophosphate could be cyclized to (+)-bornyl pyrophosphate, but the presence of high levels of phosphatases in the extract prevented an accurate assessment of substrate specificity. The competing phosphatases were removed by combination of gel filtration on Sephadex G-150, chromatography on hydroxylapatite, and chromatography on O-(diethylaminoethyl)-cellulose. In these fractionation steps, activities for the cyclization of neryl pyrophosphate and geranyl pyrophosphate to bornyl pyrophosphate were coincident, and on the removal of competing phosphatases, the synthetase was shown to prefer geranyl pyrophosphate as substrate ($\text{V}\text{K}{}_{\mathrm{m}}$ for geranyl pyrophosphate was 20-fold that of neryl pyrophosphate). No interconversion of geranyl and neryl pyrophosphates was detected. The partially purified bornyl pyrophosphate synthetase had an apparent molecular weight of 95,000, and required Mg²⁺ for catalytic activity (K_m for Mg²⁺ ~ 3.5 mm). Mn²⁺ and other divalent cations were ineffective in promoting the formation of bornyl pyrophosphate. The enzyme exhibited a pH optimum at 6.2 and was strongly inhibited by both p-hydroxymercuribenzoate and diisopropylfluorophosphate. Bornyl pyrophosphate synthetase is the first monoterpene synthetase to be isolated free from competing phosphatases, and the first to show a strong preference for geranyl pyrophosphate as substrate. A mechanism for the cyclization of geranyl pyrophosphate to bornyl pyrophosphate is proposed.     

Template-directed synthesis of oligoguanylates in the presence of metal ions

We have studied the condensation reaction of ImpG² on a poly(C) template, in the presence of various metal ions. With Mg²⁺ as co-catalyst we confirmed that Pb²⁺ and Zn²⁺ are effective catalysts. A catalytic effect was also observed for Bi³⁺, Sb³⁺ and Mn²⁺. Bi³⁺ and Sn²⁺, like Pb²⁺, favored the formation of 2′-5′ linkages. With Mn²⁺ a rather complex mixture of oligomers is formed, some of which contain pyrophosphate linkages. None of the metal ions investigated behaved like Zn²⁺ in favoring the formation of the naturally occurring 3′-5′ linkages.     

Monoterpene formation by leucoplasts of Citrofortunella mitis and Citrus unshiu

Leucoplasts of immature calamondin and satsuma fruits were incubated with [1-¹⁴C] isopentenyl pyrophosphate under various conditions. Optimal incorporation of the tracer into geranyl pyrophosphate and monoterpene hydrocarbons occurred in the presence of exogenous dimethylallyl pyrophosphate and Mn²⁺ which was more effective than Mg²⁺. The dependence of dimethylallyl pyrophosphate showed that about 10 moles were required for 1 mole of isopentenyl pyrophosphate for the best recovery in monoterpene hydrocarbon biosynthesis. A time-course incorporation of isopentenyl pyrophosphate revealed that the C₁₀ hydrocarbon elaboration was dependent on the geranyl pyrophosphate production and at no time neryl pyrophosphate was synthesized by leucoplasts. The amount of labelled farnesyl pyrophosphate was rather low whatever the conditions used in the experiments and sesquiterpene hydrocarbon biosynthesis was never observed.Abbreviations DMAPP dimethylallyl pyrophosphate - FPP farnesyl pyrophosphate - GPP geranyl pyrophosphate - IPP isopentenyl pyrophosphate - LPP linalyl pyrophosphate - NPP neryl pyrophosphate     

Prenyltransferases from the flavedo of Citrus sinensis

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 (2E,6E)- and (2Z,6E)-farnesyl pyrophosphate. The two C₁₅-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.     

Formation of coA esters of cinnamic acid derivatives by extracts of Brassica napo-brassica root tissue

An enzyme fraction from aged swede root disks catalyses the formation of CoA thioesters of cinnamic acids in the presence of CoA, ATP and Mg²⁺. The enzyme shows activity only to those cinnamic acid derivatives bearing a phenolic OH group, p-coumaric and ferulic acids being the most active substrates. The requirement for Mg²⁺ can be replaced by Mn²⁺, Co²⁺ or Ni²⁺. The requirement for ATP could not be replaced by GTP, CTP, UTP, ADP or AMP. ADP and AMP, but not pyrophosphate, inhibited the ATP dependent activation of p-coumarate. The activity was inhibited by N-ethylmaleimide and p-chloro-mercuribenzoate which suggests a requirement for -SH groups for activation. The activity of the enzyme is low in freshly prepared disks but rises during ageing, particularly if the ageing is carried out in the presence of low concentrations of ethylene.