The purification of 3,3-dimethylallyl- and geranyl-transferase and of isopentenyl pyrophosphate isomerase from pig liver

The enzyme catalysing the synthesis of farnesyl pyrophosphate from dimethylallyl pyrophosphate and isopentenyl pyrophosphate, or from geranyl pyrophosphate and isopentenyl pyrophosphate, has been purified 100-fold from homogenates of pig liver. The enzyme has optimum pH 7.9 and requires Mg(2+) as activator in preference to Mn(2+); it is inhibited by iodoacetamide, N-ethylmaleimide, p-hydroxymercuribenzoate and phosphate ions in addition to the products of the reaction, inorganic pyrophosphate and farnesyl pyrophosphate. From product-inhibition studies of the geranyltransferase reaction, the order of addition of substrates to and release of products from the enzyme has been deduced: geranyl pyrophosphate combines with the enzyme first, followed by isopentenyl pyrophosphate. Farnesyl pyrophosphate dissociates from the enzyme before inorganic pyrophosphate. The existence of isopentenyl pyrophosphate isomerase in liver is confirmed. Methods for the preparation of the pyrophosphate esters of isopentenol, 3,3-dimethylallyl alcohol, geraniol and farnesol are also described.     

Characterization of the Saccharomyces cerevisiae cis-prenyltransferase required for dolichyl phosphate biosynthesis

The prenyltransferase involved in the biosynthesis of dolichyl phosphate has been characterized in Saccharomyces cerevisiae. Although the enzyme is predominantly membrane-bound, a significant percentage was found in the soluble fraction. The prenyltransferase preferentially utilizes farnesyl pyrophosphate as the allylic substrate and isopentenyl pyrophosphate as cosubstrate with half-maximal velocities obtained at 25 and 6.7 microM, respectively. The enzymatic activity is sensitive to sulfhydryl reagents and is inhibited by all detergents tested, except 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate at concentrations less than 5 mM. The product of the reaction has been characterized as an alpha-unsaturated polyprenyl pyrophosphate, containing 12-15 isoprene units, approximately two isoprene units shorter than the endogenous yeast dolichyl phosphate. The stereochemistry of addition of isoprene units by the prenyltransferase was shown to be cis by a comparison of the HPLC retention time for a pentadecaprenyl phosphate derived from the in vitro reaction product with that for an authentic mixture of alpha-cis- and alpha-trans-pentadecaprenyl phosphates.     

Substrate Binding of avian liver prenyltransferase.

Prenyltransferase (farnesyl pyrophosphate synthetase) was purified from avian liver and characterized by Sephadex and sodium dodecyl sulfate gel chromatography, peptide mapping, and end-group analysis. The enzyme is 85 800 +/- 4280 daltons and consists of two identical subunits as judged by sodium dodecyl sulfate gel electrophoresis, peptide mapping, and end-group analysis. Chemical analysis of the protein revealed no lipid or carbohydrate components. Avian prenyltransferase synthesizes farnesyl pyrophosphate from either dimethylallyl or geranyl pyrophosphate and isopentenyl pyrophosphate. A lower rate of geranylgeranyl pyrophosphate synthesis from farnesyl pyrophosphate and isopentenyl pyrophosphate was also demonstrated. Michaelis constants for farnesyl pyrophosphate synthesis are 0.5 muM for both isopentenyl pyrophosphate and geranyl pyrophosphate. The V max for the reaction is 1990 nmol min-1 mg-1 (170 mol min-1 mol-1 enzyme). Substrate inhibition by isopentenyl pyrophosphate is evident at high isopentenyl pyrophosphate and low geranyl pyrophosphate concentrations. Michaelis constants for geranylgeranyl pyrophosphate synthesis are 9 muM for farnesyl pyrophosphate and 20 muM for isopentenyl pyrophosphate. The Vmax is 16 nmol min-1 mg-1 (1.4 mol min-1 mol-1 enzyme). Two moles of each of the allylic substrates is bound per mol of enzyme. The apparent dissociation constants for dimethylallyl, geranyl, and farnesyl pyrophosphates are 1.8, 0.17, and 0.73 muM, respectively. Dimethylallyl and geranyl pyrophosphates bound competitively to prenyltransferase with one-for-one displacement. Four moles of isopentenyl pyrophosphate was bound per mole of enzyme. Citronellyl pyrophosphate, an analogue of geranyl pyrophosphate, was competitive with the binding of 2 of the 4 mol of isopentenyl pyrophosphate bound. The data are interpreted to indicate that each subunit of avian liver prenyltransferase has a single allylic binding site accommodating dimethylallyl, geranyl, and farnesyl pyrophosphates, and one binding site for isopentenyl pyrophosphate. In the absence of an allylic pyrophosphate or analogue, isopentenyl pyrophosphate also can bind to the allylic site.     

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     

Characterization and distribution of cis-prenyl transferase participating in liver microsomal polyisoprenoid biosynthesis.

The properties of rat liver cis-prenyl transferase, mediating the synthesis of polyisoprenoid pyrophosphate from trans,trans-farnesyl pyrophosphate and [3H]isopentenyl pyrophosphate were studied. The Km values for farnesyl pyrophosphate and isopentenyl pyrophosphate were found to be 25 microM and 4.4 microM, respectively. Appropriate conditions were established to measure the condensation reaction, which was linear during the first hour using 1 mg microsomal protein. Various detergents could solubilize the enzyme, but the presence of Triton X-100 was required during the incubation to obtain full activity. There was also an absolute requirement for Mg2+ and the pH maximum was 7.0. Inorganic phosphate, especially pyrophosphate, proved to be inhibitory. cis-Prenyl transferase is associated mainly with the cytoplasmic surface of rough microsomes and, to some extent, also with smooth I microsomes, but was almost absent from smooth II microsomes. At all localizations, the product is polyprenyl pyrophosphate and to some extent, also polyprenyl monophosphate. The isoprenoids formed contain 15-18 units in the presence of detergents and 16-20 units in the absence of detergents.     

Biosynthesis of geraniol and nerol and their β-d-glucosides in Perlargonium graveolens and Rosa dilecta

1. 3R-[2-(14)C]Mevalonate was incorporated into geranyl and neryl beta-d-glucosides in petals of Rosa dilecta in up to 10.6% yield, and the terpenoid part was specifically and equivalently labelled in the moieties derived from isopentenyl pyrophosphate and 3,3-dimethylallyl pyrophosphate. A similar labelling pattern, with incorporations of 0.06-0.1% was found for geraniol or nerol formed in leaves of Pelargonium graveolens The former results provide the best available evidence for the mevalonoid route to regular monoterpenes in higher plants. 2. Incorporation studies with 3RS-[2-(14)C,(4R)-4-(3)H(1)]-mevalonate and its (4S)-isomer showed that the pro-4R hydrogen atom of the precursor was retained and the pro-4S hydrogen atom was eliminated in both alcohols and both glucosides. These results suggest that the correlation of retention of the pro-4S hydrogen atom of mevalonate with formation of a cis-substituted double bond, such as has been found in certain higher terpenoids, does not apply to the biosynthesis of monoterpenes. It is proposed that either nerol is derived from isomerization of geraniol or the two alcohols are directly formed by different prenyltransferases. Possible mechanisms for these processes are discussed. 3. The experiments with [(14)C,(3)H]mevalonate also show that in these higher plants, as has been previously found in animal tissue and yeast, the pro-4S hydrogen atom of mevalonate was lost in the conversion of isopentenyl pyrophosphate into 3,3-dimethylallyl pyrophosphate.     

The Enzymatic Synthesis of Rubber Polymer in Parthenium argentatum Gray

Washed rubber particles isolated from stem homogenates of Parthenium argentatum Gray by ultracentrifugation and gel filtration on columns of LKB Ultrogel AcA34 contain rubber transferase which catalyzes the polymerization of isopentenyl pyrophosphate into rubber polymer. The polymerization reaction requires Mg²⁺ isopentenyl pyrophosphate, and an allylic pyrophosphate. The K_m values for Mg²⁺, isopentenyl pyrophosphate, and dimethylallyl pyrophosphate were 5.2 × 10⁻⁴ molar, 8.3 × 10⁻⁵ molar, and 9.6 × 10⁻⁵ molar, respectively. The molecular characteristics of the rubber polymer synthesized from [¹⁴C]isopentenyl pyrophosphate were examined by gel permeation chromatography on three linear columns of 1 × 10⁶ to 500 Ångstroms Ultrastyragel in a Waters 150C Gel Permeation Chromatograph. The peak molecular weight of the radioactive polymer increased from 70,000 in 15 minutes to 750,000 in 3 hours. The weight average molecular weight of the polymer synthesized over a 3 hour period was 1.17 × 10⁶ compared to 1.49 × 10⁶ for the natural rubber polymer extracted from the rubber particles. Over 90% of the in vitro formation of the rubber polymer was de novo from dimethylallyl pyrophosphate and isopentenyl pyrophosphate. Treatment of the washed rubber particles with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate solubilized the rubber transferase. The solubilized enzyme(s) catalyzed the polymerization of isopentenyl pyrophosphate into rubber polymer with a peak molecular weight of 1 × 10⁵ after 3 hours of incubation with Mg²⁺ and dimethylallyl pyrophosphate. The data support the conclusion that the soluble preparation of rubber transferase is capable of catalyzing the formation of a high molecular weight rubber polymer from an allylic pyrophosphate initiator and isopentenyl pyrophosphate monomer.     

Studies on the regulation of glycoprotein biosynthesis. An investigation of the rate-limiting steps of dolichyl phosphate biosynthesis.

The possible role of HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase (the rate-controlling enzyme of cholesterol biosynthesis) in regulating the rate of dolichyl phosphate biosynthesis in rat liver was investigated. Rats were either fasted 48 h or fed diets supplemented with the drug cholestyramine. The activity of HMG-CoA reductase was 5000-fold greater in liver from cholestyramine-fed rats as compared to fasted rats. The activity of dolichyl phosphate synthetase, the prenyl transferase responsible for the biosynthesis of dolichyl phosphate from farnesyl pyrophosphate and isopentenyl pyrophosphate, was similar in both nutritional conditions and was markedly less active than HMG-CoA reductase even in the fasted state. Acetate incorporation into cholesterol was 2200-fold greater in liver slices from cholestyramine-fed rats as compared to fasted rats. By contrast, acetate incorporation into dolichyl phosphate was only 6-fold higher. Further studies suggested that the levels of farnesyl pyrophosphate and isopentenyl pyrophosphate are several hundred-fold greater in liver from cholestyramine-treated rats. From these results, it is concluded that the rate of dolichyl phosphate biosynthesis in rat liver is not regulated by the activity of HMG-CoA reductase but is probably regulated at the level of dolichyl phosphate synthetase.     

Aspects of the stereochemistry of torularhodin biosynthesis

1. The incorporation of [2-(14)C]acetate, [2-(14)C]mevalonic acid and [2-(14)C,2-(3)H(2)]-mevalonic acid into torulene and torularhodin by Rhodotorula rubra and Rhodotorula glutinis was studied. 2. A recovery of 14.3% of the label was obtained on decarboxylation of the torularhodin biosynthesized from [2-(14)C]mevalonic acid. 3. An analysis of the (3)H/(14)C ratio in torularhodin gave a value of 9.44:8. 4. These results, obtained by different experimental techniques, show that the reactions in the conversion of the dimethyl group of isopentenyl pyrophosphate into the 16',17'-position of torularhodin must be free from randomization. A mechanism for the isomerization of isopentenyl pyrophosphate to dimethylallyl pyrophosphate is suggested.     

Isolation and characterization of MOD5, a gene required for isopentenylation of cytoplasmic and mitochondrial tRNAs of Saccharomyces cerevisiae.

The mod5-1 mutation is a nuclear mutation in Saccharomyces cerevisiae that reduces the biosynthesis of N6-(delta 2-isopentenyl)adenosine in both cytoplasmic and mitochondrial tRNAs to less than 1.5% of wild-type levels. The tRNA modification enzyme, delta 2-isopentenyl pyrophosphate:tRNA isopentenyl transferase, cannot be detected in vitro with extracts from mod5-1 cells. A characterization of the MOD5 gene would help to determine how the same enzyme activity in different cellular compartments can be abolished by a single nuclear mutation. To that end we have cloned the MOD5 gene and shown that it restores delta 2-isopentenyl pyrophosphate:tRNA isopentenyl transferase activity and N6-(delta 2-isopentenyl)adenosine to tRNA in both the mitochondria and the nucleus/cytoplasm compartments of mod5-1 yeast cells. That MOD5 sequences are expressed in Escherichia coli and can complement an N6-(delta 2-isopentenyl)-2-methylthioadenosine-deficient E. coli mutant leads us to conclude that MOD5 is the structural gene for delta 2-isopentenyl pyrophosphate:tRNA isopentenyl transferase.     

Prenyltransferase: the mechanism of the reaction.

The enzyme, prenyltransferase, which normally catalyzes the addition of an allylic pyrophosphate to isopentenyl pyrophosphate, has been found to catalyze the hydrolysis of its allylic substrate. The rate of this hydrolysis is markedly stimulated by inorganic pyrophosphate. Competition experiments with 2-fluoroisopentenyl pyrophosphate and inorganic pyrophosphate demonstrated that inorganic pyrophosphate stimulated hydrolysis by binding at the isopentenyl pyrophosphate site. Hydrolysis carried out in H218O or with (1S)-[1-3H]geranyl pyrophosphate show the C-O bond is broken and the C1 carbon of geranyl pyrophosphate is inverted in the process. These results are interpreted to favor a carbonium ion mechanism for the prenyltransferase reaction.     

Heptaprenyl pyrophosphate synthetase from Bacillus subtilis

Heptaprenyl pyrophosphate synthetase was detected in partially purified extracts of Bacillus subtilis. The enzyme catalyzed the synthesis of all-trans C35 prenyl pyrophosphate from isopentenyl pyrophosphate and farnesyl or geranylgeranyl pyrophosphate, but it did not catalyze a reaction between isopentenyl pyrophosphate and either dimethylallyl or geranyl pyrophosphate. The enzyme reaction proceeded with an elimination of 2-pro-R hydrogen of isopentenyl pyrophosphate without accumulation of any prenyl pyrophosphate shorter than C35. The molecular weight of the enzyme was estimated by gel filtration to be 45,000. Michaelis constants for isopentenyl, farnesyl, and geranylgeranyl pyrophosphate were 12.8, 13.3, and 8.3 microM, respectively.     

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.     

Studies on the biosynthesis of dolichyl phosphate: Evidence for the in vitro formation of 2,3-dehydrodolichyl phosphate

A particulate enzyme preparation from hen oviduct is shown to carry out the biosynthesis of a long chain polyprenyl phosphate from isopentenyl pyrophosphate and farnesyl pyrophosphate. The compound has the physical and chemical properties of 2,3-dehydrodolichyl phosphate. The enzyme system is inhibited by EDTA and stimulated by Triton X-100 and dithiothreitol. If the product of the reaction is 2,3-dehydrodolichyl phosphate, it may be derived from 2,3-dehydrodolichyl pyrophosphate, a likely intermediate in the biosynthesis of dolichyl phosphate.     

Insight into the activation mechanism of Escherichia coli octaprenyl pyrophosphate synthase derived from pre-steady-state kinetic analysis

Octaprenyl pyrophosphate synthase (OPPs) catalyzes the sequential condensation of five molecules of isopentenyl pyrophosphate with farnesyl pyrophosphate to generate all-trans C40-octaprenyl pyrophosphate, which constitutes the side chain of ubiquinone. Due to the slow product release, a long-chain polyprenyl pyrophosphate synthase often requires detergent or another factor for optimal activity. Our previous studies in examining the activity enhancement of Escherichia coli undecaprenyl pyrophosphate synthase have demonstrated a switch of the rate-determining step from product release to isopentenyl pyrophosphate (IPP) condensation reaction in the presence of Triton [12]. In order to understand the mechanism of enzyme activation for E. coli OPPs, a single-turnover reaction was performed and the measured IPP condensation rate (2 s(-1)) was 100 times larger than the steady-state rate (0.02 s(-1)). The high molecular weight fractions and Triton could accelerate the steady-state rate by 3-fold (0.06 s(-1)) but insufficient to cause full activation (100-fold). A burst product formation was observed in enzyme multiple turnovers indicating a slow product release.     

Isoprenoid enzyme systems of silkworm. II. Formation of the juvenile hormone skeletons by farnesyl pyrophosphate synthetase II

Comparative substrate specificities of farnesyl pyrophosphate synthetases I and II purified from larvae of silkworm, Bombyx mori, were studied by use of the possible biosynthetic intermediates of juvenile hormones in the insect. In the presence of Mn2+ ions farnesyl pyrophosphate synthetase II showed higher activity than synthetase I and the corresponding enzyme from pig liver with the following substrate homologues: (Z)-3-methyl-2-pentenyl-, 3-ethyl-3-butenyl-, (2E,6Z)-3,7-dimethyl-2,6-nonadienyl-, and (2E,6Z)-3-ethyl-7-methyl-2,6-nonadienyl pyrophosphate. When (Z)-3-methyl-2-pentenyl-, 3-ethyl-3-butenyl-, and isopentenyl pyrophosphate were mixed and incubated with farnesyl pyrophosphate synthetase II, (2E,6E,10Z)-3,11-dimethyl-7-ethyl-2,6,10-tridecatrienyl-, (2E,6E,10Z)-3,7,11-trimethyl-2,6,10-tridecatrienyl, and a trace amount of (2E,6E,10Z)-3,7-diethyl-11-methyl-2,6,10-tridecatrienyl pyrophosphate, whose carbon skeletons were the same as those of juvenile hormone I, II, and O, respectively, were formed. (Z)-3-Methyl-2-pentenyl pyrophosphate was produced from 3-ethyl-3-butenyl pyrophosphate as a single product by the action of silkworm isopentenyl pyrophosphate isomerase, though the enzyme activity was much lower with this substrate than with the usual substrate, isopentenyl pyrophosphate.     

Phytoene synthase and phytoene dehydrogenase associated with envelope membranes from spinach chloroplasts

Envelope membranes of spinach chloroplasts contain appreciable activities of the carotenogenic enzymes phytoene synthase (formation of phytoene by condensation of two molecules geranylgeranyl pyrophosphate) and phytoene dehydrogenase (formation of lycopene from phytoene), plus a phosphatase activity. These results were obtained by coincubation experiments using isolated envelope membranes and either a phytoene-forming in vitro system (from [1-¹⁴C]isopentenyl pyrophosphate) or [¹⁴C]geranylgeranyl pyrophosphate or a geranylgeranyl-pyrophosphate-forming in vitro system (from [1-¹⁴C]isopentenyl pyrophosphate). Within thylakoids carotenogenic enzymes could not be detected. It is concluded that the chloroplast envelope is at least a principal site of the membrane-bound steps of carotenoid biosynthesis in chloroplasts.Abbreviastions Chlorophyll a_GC Chlorophyll a, esterified with geranylgeraniol - GGPP geranylgeranyl pyrophosphate - HPLC high pressure liquid chromatography - IPP isopentenyl pyrophosphate     

Synthesis of 10,11-dihydrofarnesyl pyrophosphate from 6,7-dihydrogeranyl pyrophosphate by prenyltransferase

The syntheses of 6,7-dihydrogeraniol and of its pyrophosphate are described. It is shown that this analogue of geranyl pyrophosphate is a substrate for liver prenyltransferase and that the product synthesized by this enzyme from it and isopentenyl pyrophosphate is 10,11-dihydrofarnesyl pyrophosphate. The K(m) value for 6,7-dihydrogeranyl pyrophosphate was determined to be 1.11+/-0.19mum as compared with 4.34+/-1.71mum for geranyl pyrophosphate. The maximum reaction velocity with the artifical substrate was, however, only about one-fourth of that observed with geranyl pyrophosphate. The binding of isopentenyl pyrophosphate to the enzyme was not affected by the artificial substrate.     

Isopentenyl pyrophosphate isomerase from liver

Isopentenyl pyrophosphate isomerase (EC 5.3.3.2) was purified from extracts of pig liver by ammonium sulphate fractionation and by gel filtration. After about 20-fold purification the preparations were free of phosphatase and prenyltransferase (EC 2.5.1.1), the two enzymes that could have interfered with the assays. The isomerase has a distinct pH optimum at 6.0 and is activated by Mn(2+) in preference to Mg(2+). The K(m) value for isopentenyl pyrophosphate is 4x10(-6)m. The equilibrium of the reaction favours the formation of dimethylallyl pyrophosphate. The reversibility of the isomerase reaction was demonstrated directly by the formation of isopentenyl pyrophosphate from dimethylallyl pyrophosphate. It is suggested that two prenyl isomerases might exist, one involved in the synthesis of trans- and another in the synthesis of cis-polyprenyl substances.     

Preparation of 2-amino-2-deoxy-α-D-glucopyranosyl phosphate from uridine 5'-(2-acetamido-2-deoxy-α-D-glucopyranosyl pyrophosphate)

Hydrazine treatment of uridine 5'-(2-acetamido-2-deoxy-α-D-glucopyranosyl pyrophosphate) for 1 h resulted in N-deacetylation and cleavage of the pyrophosphate bond to give 2-amino-2-deoxy-α-D-glucopyranosyl phosphate as the main compound. It was separated from other degradation products by paper electrophoresis and isolated in a yield of 50–60%.