Studies on isoprenoid biosynthesis with bacterial intact cells

For the study on the regulation of isoprenoid biosynthesis with intact cells, some strains of bacteria capable of growing on mevalonate as a sole carbon source were isolated from soil. Many of them incorporated [¹⁴C]-mevalonate, [¹⁴C]isopentenyl- and [¹⁴C]farnesyl pyrophosphates into the cells. However, radioactivity was found in their degradation products but not in isoprenoids. Addition of [¹⁴C]isopentenyl pyrophosphate, farnesyl pyrophosphate and Mg²⁺ ions in combination to the culture of a strain of Arthrobacter gave rise to ¹⁴C-incorporation into isoprenoids. Radioactivity was found in polyprenol, its pyrophosphate, monophosphate and fatty acid esters. The reactions of isopentenyl- and farnesyl pyrophosphates syntheses seemed to be rate-limiting steps.     

Biosynthesis of mono- and sesquiterpenes in peppermint from mevalonate-2-C

The volatile oil from peppermint (Mentha piperita L.) is composed primarily of monoterpenes with less than 2% sesquiterpenes. However, radioactivity from mevalonate-2-¹⁴C is incorporated into caryophyllene and other sesquiterpene hydrocarbons much more extensively than into monoterpenes by peppermint cuttings. Both mono- and sesquiterpenes show maximum incorporation of label after 6 hr (0·03% vs. 0·33% of the physiological isomer) and lose 75% of the incorporated label after an additional 6 hr. Caryophyllene derived from mevalonate-2-¹⁴C after 6 hr of incorporation was chemically degraded. The isoprenoid origin of caryophyllene was confirmed, and preferential labelling of the isopentenyl pyrophosphate derived portions of the molecule was noted. On the basis of such evidence it appears that separate sites may exist for the biosynthesis of mono- and sesquiterpenes and that an endogenous dimethylallyl pyrophosphate pool may participate in the biosynthesis of sesquiterpenes in peppermint.     

Partial purification and properties of prenyltransferase from Pisum sativum

Prenyltransferase (EC 2.5.1.1; assayed as farnesyl pyrophosphate synthetase)was purified 106-fold from an homogenate of 3-day-old seedlings of Pisum sativum. Some of the properties of the purified enzyme were determined and these differed in several significant respects from those reported for preparations from other sources, e.g. the apparent MW was 96000 ± 4000 and the preparation could be dissociated into two subunits of MW 45000 ± 3000. The total activity of the extractable enzyme went through a sharp maximum (in the range 1 to 28 days) 3 days after germination. Farnesyl pyrophosphate was formed in cell-free extracts of peas from either isopentenyl pyrophosphate alone, or this together with geranyl pyrophosphate (optimum yields 1.2 and 10% respectively). Use of [1-¹⁴C]- and [4-¹⁴C]-isopentenyl pyrophosphates as the sole substrates and degradation of the products showed that the crude extracts contained a pool of the biogenetic equivalent of 3,3-dimethylallyl pyrophosphate. No analogous pool of geranyl pyrophosphate could be detected.     

异戊烯基焦磷酸转运对雷公藤甲素生物合成的影响

雷公藤甲素是一种具有显著抗炎、抗肿瘤和免疫抑制活性的天然产物,市场需求量大,临床应用前景广阔.文中以雷公藤悬浮细胞为实验材料,通过对不同培养时期(7d、14 d)的细胞外源性添加D,L-甘油醛(DLG)以阻断异戊烯基焦磷酸(IPP)转运,分析诱导前后的细胞活性及生物量、雷公藤甲素累积量及其生物合成上游途径关键酶基因的变...     

Membrane-bound geranylgeranyl diphosphate phosphatases: purification and characterization from Croton stellatopilosus leaves

Geranylgeranyl diphosphate phosphatase is an enzyme catalyzing the dephosphorylation of geranylgeranyl diphosphate (GGPP) to form geranylgeraniol (GGOH). The enzyme activity of GGPP phosphatase was detected in leaves of Croton stellatopilosus, a Thai medicinal plant containing plaunotol, a commercial anti-peptic acyclic diterpenoid. Enzymological studies of GGPP phosphatase in C. stellatopilosis leaves revealed that the enzyme is a membrane-bound protein that could be removed from 20,000g pellet by 0.1% Triton X-100 without significant loss of enzyme activity. The solubilized enzyme preparation was separated into two activity peaks, PI and PII, by BioGel A gel filtration chromatography. PI and PII were both partially purified and characterized. PI appeared to be a tetrameric enzyme with its native molecular mass of 232kDa and subunit size of 58kDa, whereas PII was a monomeric enzyme with a molecular mass of 30-34kDa. Both phosphatases utilized GGPP as the preferred substrate over farnesyl and geranyl diphosphates. The apparent K(m) values for GGPP of PI and PII appeared to be 0.2 and 0.1mM, respectively. Both activities were Mg(2+) independent and exhibited slightly acidic pH optima, 6.0-6.5 for PI and 6.5-7.0 for PII. The catalytic activities of PII was strongly inhibited by 1.0mM of Zn(2+), Mn(2+) and Co(2+), whereas that of PI was not affected. Both enzyme preparations were very stable upon storage at -20 degrees C for 45 days without significant loss of phosphatase activity. The presence of GGPP phosphatase enzymes in C. stellatopilosus is consistent with its putative involvement in the biosynthetic pathway of plaunotol although whether PI or PII is the actual enzyme involved in the pathway remains to be clarified.     

Isolation of intact plastids from a range of plant tissues

A technique for the isolation of intact plastids from spinach (Spinacia oleracea) and pea (Pisum sativum) leaves, pea roots and castor bean (Ricinus communis) endosperm is described. This technique involves brief centrifugation of whole homogenates on density gradients. Intact plastids were located in the gradient by assaying for triose phosphate isomerase activity. Contamination of the plastic peak with mitochondria and microbodies was estimated by measurement of cytochrome oxidase and catalase, respectively. For three of the four tissues the level of contamination of the plastids by these organelles was 2% or less. The sedimentation behavior of microbodies from different tissues is discussed.     

Biosynthesis of monoterpenes in plants from 14C-labelled acetate and CO2

Degradation of (+)-isothujone biosynthesized by Tanacetum vulgare or Thuja plicata from acetate-[1-¹⁴C], -[2-¹⁴C] and -[2-³H₃] or from CO₂-[14C] at physiological concentration revealed a pattern of asymmetric labelling whereby tracer predominantly (72–98% resided in that part of the skeleton derived from IPP. This is similar to the patterns previously obtained for uptake of MVA-[2-¹⁴C] but differed from those reported in other species with acetate-[¹⁴C] as precursor. Within the IPP-derived moiety the 3 parts derived from acetate units were not equivalently labelled. Partial degradations of geraniol and (+)-pulegone formed in Pelargonium graveolens and Mentha pulegium after uptake of ¹⁴C-labelled acetate or CO₂ showed that the C-2 units of the skeletons of these monoterpenes were also labelled to widely differing extents and these patterns persisted over a range of feeding and seasonal conditions. These results suggest that metabolic pools of acetyl-CoA and/or acetoacetyl-CoA exist in these plants. The general occurrence of such pools and the consequent nonequivalent labelling patterns in secondary metabolism could invalidate biosynthetic conclusions drawn from partial degradations of labelled natural products.     

Adenylosuccinate synthetase and adenylosuccinate lyase from plant tissues

1. Enzymes that convert IMP into adenylosuccinate (adenylosuccinate synthetase) and adenylosuccinate into AMP (adenylosuccinate lyase) were isolated from wheat germ and pea seeds and their properties are described. 2. These enzymes were purified approx. 200-fold from wheat-germ extracts. 3. A heat treatment provided adenylosuccinate lyase free of adenylosuccinate synthetase but the behaviour of the two enzymes was almost identical in a number of fractionation procedures. The two activities were finally separated by filtration on Sephadex G-100. 4. The identification of these enzymes in plant tissues is discussed in relation to the pathway of purine synthesis.     

Metabolism of mevalonic acid to phosphorylated intermediates in a cell-free extract from Nepeta cataria leaves

A cell-free extract has been prepared from leaves of Nepeta cataria plants which converts mevalonic acid (MVA) to mevalonic acid phosphate (MVAP), mevalonic acid pyrophosphate (MVAPP) and isopentenylpyrophosphate (IPP). These enzymes are found in the 30 000 g supernatant. The activities are maximal at pH 7 and the formation of mevalonic acid pyrophosphate and isopentenyl-pyrophosphate reaches a maximal level after an incubation time of 180 min whereas the level of mevalonic acid phosphate begins to decrease after 90 min.     

Invitro biosynthesis of isoprene from mevalonate utilizing a rat liver cytosolic fraction

The $\text{in}\text{vitro}$ biosynthesis of isoprene from DL-mevalonate in the cytosolic fraction of rat liver is described. Evidence is provided suggesting a non-enzymatic formation of isoprene from isopentenyl pyrophosphate and/or dimethylallyl pyrophosphate. Furthermore, the data establish an alternate fate of the mevalonate carbon skeleton providing the first evidence that breath isoprene is linked to cholesterol biosynthesis.     

Effect of gibberellic acid on mevalonate activation in germinating Corylus avellana seeds

Giberellic acid (GA) induced germination of hazel seeds is accompanied by early increases in the specific and total activities of MVA kinase in the embryonic axes. This is followed by an increase in the activity of decarboxylation of MVA by the whole axes. The activity of MVA kinase in the cotyledons is not affected by GA treatment although increased uptake of MVA results in increased decarboxylation by cotyledon slices. The effects of cofactors and inhibitors on the activities of MVA kinase and MVA decarboxylation in a cell free extract of hazel cotyledons are described.     

Substrate binding mode and reaction mechanism of undecaprenyl pyrophosphate synthase deduced from crystallographic studies

Undecaprenyl pyrophosphate synthase (UPPs) catalyzes eight consecutive condensation reactions of farnesyl pyrophosphate (FPP) with isopentenyl pyrophosphate (IPP) to form a 55-carbon long-chain product. We previously reported the crystal structure of the apo-enzyme from Escherichia coli and the structure of UPPs in complex with sulfate ions (resembling pyrophosphate of substrate), Mg(2+), and two Triton molecules (product-like). In the present study, FPP substrate was soaked into the UPPs crystals, and the complex structure was solved. Based on the crystal structure, the pyrophosphate head group of FPP is bound to the backbone NHs of Gly29 and Arg30 as well as the side chains of Asn28, Arg30, and Arg39 through hydrogen bonds. His43 is close to the C2 carbon of FPP and may stabilize the farnesyl cation intermediate during catalysis. The hydrocarbon moiety of FPP is bound with hydrophobic amino acids including Leu85, Leu88, and Phe89, located on the alpha3 helix. The binding mode of FPP in cis-type UPPs is apparently different from that of trans-type and many other prenyltransferases which utilize Asprich motifs for substrate binding via Mg(2+). The new structure provides a plausible mechanism for the catalysis of UPPs.     

The first prenylation step in hyperforin biosynthesis

Prenylation reactions contribute considerably to the diversity of natural products. Polyprenylated secondary metabolites include hyperforin which is both quantitatively and pharmacologically a major constituent of the medicinal plant Hypericum perforatum (St. John's wort). Cell cultures of the related species Hypericum calycinum were found to contain a prenyltransferase activity which is likely to catalyze the first prenylation step in hyperforin biosynthesis. The enzyme was soluble and dependent on a divalent cation, with Fe2+ leading to maximum activity (Km=3.8 mM). The preferred prenyl donor was DMAPP (Km=0.46 mM) and the preferred prenyl acceptor was phlorisobutyrophenone (Km=0.52 mM). A broad pH optimum from 6.5 to 8.5 and a temperature optimum from 35 to 40 degrees C were observed. The formation of hyperforins in H. calycinum cell cultures was preceded by an increase in dimethylallyltransferase activity, with the maximum specific activity being 3.6 microkat/kg protein.     

Evidence for geraniol as an obligatory precursor of isothujone

Isothujone (trans-thujan-3-one) was formed from MVA-[¹⁴C, ³H] in Tanacetum vulgare with retention of the pro-(4R) hydrogen of precursor, but with loss of the pro-(4S) hydrogen and of one hydrogen from C-5. Cell-free extracts could not sustain the formation of isothujone from MVA but yielded geraniol and nerol (3,7-dimethylocta-trans-2,6-dien-1-ol and its cis isomer) with retention of the pro-(4R) and loss of the pro-(4S) hydrogen in each case: no hydrogen was lost from C-5 of MVA in formation of geraniol, but one such atom was lost in the formation of nerol. These results support the sequence: geraniol → nerol → isothujone: in which the first two compounds (or their biogenetic equivalents) are interconverted by a redox process involving their derived aldehydes. They are not consistent with a direct pathway to nerol from C₅ intermediates or with routes involving cyclisation of linalol (3,7-dimethylocta-1,6-dien-3-ol) formed directly from the C₅ compounds or from geraniol. The cell-free preparations could not interconvert geraniol and nerol, their phosphates or pyrophosphates. This may be due to the inability of a prenyltransferase-isomerase multi-enzyme system to accept exogenously-supplied intermediates under these (in vitro) conditions.     

The crystal structure of human isopentenyl diphosphate isomerase at 1.7 A resolution reveals its catalytic mechanism in isoprenoid biosynthesis

The essential Saccharomyces cerevisiae pre-messenger RNA splicing protein 24 (Prp24) has four RNA recognition motifs (RRMs) and facilitates U6 RNA base-pairing with U4 RNA during spliceosome assembly. Prp24 is a component of the free U6 small nuclear ribonucleoprotein particle (snRNP) but not the U4/U6 bi-snRNP, and so is thought to be displaced from U6 by U4/U6 base-pairing. The interaction partners of each of the four RRMs of Prp24 and how these interactions direct U4/U6 pairing are not known. Here we report the crystal structure of the first three RRMs and the solution structure of the first two RRMs of Prp24. Strikingly, RRM 2 forms extensive inter-domain contacts with RRMs 1 and 3. These contacts occupy much of the canonical RNA-binding faces (beta-sheets) of RRMs 1 and 2, but leave the beta-sheet of RRM 3 exposed. Previously identified substitutions in Prp24 that suppress mutations in U4 and U6 spliceosomal RNAs cluster primarily in the beta-sheet of RRM 3, but also in a conserved loop of RRM 2. RNA binding assays and chemical shift mapping indicate that a large basic patch evident on the surface of RRMs 1 and 2 is part of a high affinity U6 RNA binding site. Our results suggest that Prp24 binds free U6 RNA primarily with RRMs 1 and 2, which may remodel the U6 secondary structure. The beta-sheet of RRM 3 then influences U4/U6 pairing through interaction with an unidentified ligand.     

Stimulation of rat liver 4-hydroxybenzoate: polyprenyl transferase activity by a cytosolic protein factor; evidence for a polyprenyl pyrophosphate transport protein

Rat liver postmicrosomal supernatant contains a factor which stimulates the 4-hydroxybenzoate:polyprenyl transferase activity of whole mitochondria and inner mitochondrial membrane fragments. The factor involved appears to be a heat stable, nondializable protein sensitive to tryptic hydrolysis and has been partially purified. Since this protein binds nonaprenyl pyrophosphate and stimulates its transport into mitochondria but shows no similar effect with 4-hydroxybenzoate, it is suggested that this protein also acts as an all trans polyprenyl pyrophosphate carrier protein.     

Biosynthesis of irregular monoterpenes in extracts from higher plants

Extracts from Artemisia annua and Santolina chamaecyparissus converted ¹⁴C-labelled IPP, DMAPP and DMVC into artemisia ketone, its corresponding alcohol, lavandulol and trans-chrysanthemyl alcohol with up to 12.0 % incorporation of tracer. DMVC was the most effective precursor under standard conditions and led to unequal distribution of tracer in the C-5 moieties. The same extracts interconverted cis and trans-chrysanthemyl alcohols and their pyrophosphates, artemisia ketone, and artemisyl alcohol in up to 10·4% yields, but geraniol, nerol and linalol or their pyrophosphates were not precursors of any of these compounds. Formation of artemisia ketone and its alcohol from C-5 intermediates was enhanced by NAD⁺ and NADP⁺ but was unaffected by absence of oxygen. These co-factors did not affect the yields of lavandulol or trans-chrysanthemyl alcohol. These observations suggest closely related biogenetic pathways to the three irregular skeltons that do not involve the usual C-10 intermediates of monoterpene biosynthesis: i.e. the biogenetic isoprene rule is not obeyed.     

Biosynthesis of monoterpenes by Ceratocystis moniliformis

Ceratocystis moniliformis produced and excreted monoterpenes when grown on potato-dextrose broth. Geraniol, nerol, citronellol, linalol, α-terpineol, geranial and neral were identified by GC-MS. Their production commenced with the depletion of nitrogen in the growth medium and their combined concentration peaked at about 50 μg/ml on the 5th day of growth. The pathway for the biosynthesis of the identified monoterpenes was studied by supplying the radioactive precursors mevalonic acid-[2-¹⁴C], l-leucine-[4,5-³H(N)], and acetate- [2-¹⁴C] to C. moniliformis. For each precursor, the extent of incorporation into the above monoterpenes and the distribution of radioactivity in geraniol was determined. It was concluded that monoterpenes were formed via the mevalonate pathway, previously established for higher terpenes in other organisms. This represents the first information available on the biosynthetic pathway for free monoterpenes in a microbial system.     

Phosphoribosyl pyrophosphate and the measurement on inorganic pyrophosphate in plant tissues

This work provides further evidence that plants contain appreciable amounts of inorganic pyrophosphate (PPi), and that breakdown of phosphoribosyl pyrophosphate (PPRibP) does not contribute significantly to the PPi detected in plant extracts. Inorganic pyrophosphate in extracts of the roots of Pisum sativum L., clubs of the spadices of Arum maculatum L., and the developing endosperm of Zea mays L. was assayed with pyrophosphate fructose 6-phosphate 1-phosphotransferase (EC 2.7.1.90), and with sulphate adenyltransferase (EC 2.7.7.4). The two different assays gave the same value for PPi content, and for recovery of added PPi. It was shown that PPRibP is converted to PPi during the extraction of PPi. However, the amounts of PPRibP in clubs of A. maculatum and the developing endosperm of Z. mays were negligible in comparison with the contents of PPi.Abbreviations EDTA ethylenediaminetetraacetic acid - PFK(PPi) pyrophosphate fructose 6-phosphate 1-phosphotransferase - PPi inorganic pyrophosphate - PPRibP phosphoribosyl pyrophosphate