Participation of Mevalonate in the Biosynthetic Pathway of Ipomeamarone

1. The incorporation of mevalonate-2-¹⁴C into ipomeamarone in sweet potato root tissue infected by Ceratocystis fimbriata was demonstrated, but the rate was low when compared with acetate-2-¹⁴C. No dilution effect of mevalonate was noted during the incorporation of acetate-2-¹⁴C into ipomeamarone. This is very likely to result from the passive transfer of mevalonate into the cells.

2. No dilution effect of acetate during the incorporation of mevalonate-2-¹⁴C into ipomeamarone was noted. This indicates that mevalonate is not incorporated into ipomeamarone after its conversion to acetate.

3. Evidence for incorporation of acetate-2-¹⁴C into mevalonate was shown by the fact that the specific radioactivity of mevalonic acid benzhydrylamide was not lowered throughout repetitive crystallizations. These data also support the participation of mevalonate in ipomeamarone synthesis as an intermediate.

     

Participation of farnesol in the biosynthesis of ipomeamarone

Farnesol-2-¹⁴C was readily incorporated into ipomeamarone, oneof the furanoterpenoids produced in sweet potato infected withthe black rot fungus, Ceratocystis fumbriata. This was demonstratedby isolating labeled ipomeamarone and analyzing its radioauto-gramby silica gel thin layer chromatography of the extracts solublein chloroformmethanol (1: 1 , v/v), after farnesol-2-¹⁴C feedingto the tissue. Further proof for farnesol-2-¹⁴C incorporationinto ipomeamarone comes from the fact that the specific radioactivityof ipomeamarone semicarbazone was constant throughout the crystallizations.Fractionation of the label of farnesol-2-¹⁴C showed that radioactivitywas little distributed in the methanol-water fraction and wasmainly incorporated into ipomeamarone. Accordingly, it is notlikely that farnesol is incorporated into ipomeamarone afterits degradation to a small molecule(s) such as acetate. An additionalexperiment indicatedthat the incorporation of farnesol-2-¹⁴Cinto ipomeamarone markedly decreased under strict anaerobicconditions. This shows that some oxidative reactions are involvedin ipomeamarone biosynthesis from farnesol. ¹ This paper constitutes Part 91 of the Phytopathological Chemistryof Sweet Potato with Black Rot and Injury. (Received February 3, 1971; )     

Dehydroipomeamarone as an Intermediate in the Biosynthesis of Ipomeamarone, a Phytoalexin from Sweet Potato Root Infected with Ceratocystis fimbriata

Recently, we isolated dehydroipomeamarone, a new sesquiterpenoid from sweet potato (Ipomoea batatas Lam.) root tissue infected with Ceratocystis fimbriata (Ell. et Halst.). The purpose of this investigation was to determine whether dehydroipomeamarone was a precursor in the biosynthetic pathway of ipomeamarone. The incorporation of acetate-2-¹⁴C into ipomeamarone was markedly inhibited by the presence of dehydroipomeamarone. Radioactive dehydroipomeamarone was efficiently converted into ipomeamarone, and the compound was biosynthesized earlier than ipomeamarone according to a time course analysis of the production of the terpenoid. These results support the notion that dehydroipomeamarone is an immediate precursor of ipomeamarone. On the other hand, the production of ipomeamarone was slightly lessened in the presence of dehydroipomeamarone. Thus, the marked reduction of acetate-2-¹⁴C incorporation into ipomeamarone by dehydroipomeamarone may result from both isotopic dilution and an inhibitory effect by exogenous dehydroipomeamarone.     

Purine and pyrimidine metabolism in cultured white spruce (Picea glauca) cells: Metabolic fate of 14C-labeled precursors and activity of key enzymes

In order to examine the biosynthesis, interconversion, and degradation of purine and pyrimidine nucleotides in white spruce cells, radiolabeled adenine, adenosine, inosine, uracil, uridine, and orotic acid were supplied exogenously to the cells and the overall metabolism of these compounds was monitored. [8‐¹⁴C]adenine and [8‐¹⁴C]adenosine were metabolized to adenylates and part of the adenylates were converted to guanylates and incorporated into both adenine and guanine bases of nucleic acids. A small amount of [8‐¹⁴C]inosine was converted into nucleotides and incorporated into both adenine and guanine bases of nucleic acids. High adenosine kinase and adenine phosphoribosyltransferase activities in the extract suggested that adenosine and adenine were converted to AMP by these enzymes. No adenosine nucleosidase activity was detected. Inosine was apparently converted to AMP by inosine kinase and/or a non‐specific nucleoside phosphotransferase. The radioactivity of [8‐¹⁴C]adenosine, [8‐¹⁴C]adenine, and [8‐¹⁴C]inosine was also detected in ureide, especially allantoic acid, and CO₂. Among these 3 precursors, the radioactivity from [8‐¹⁴C]inosine was predominantly incorporated into CO₂. These results suggest the operation of a conventional degradation pathway. Both [2‐¹⁴C]uracil and [2‐¹⁴C]uridine were converted to uridine nucleotides and incorporated into uracil and cytosine bases of nucleic acids. The salvage enzymes, uridine kinase and uracil phosphoribosyltransferase, were detected in white spruce extracts. [6‐¹⁴C]orotic acid, an intermediate of the de novo pyrimidine biosynthesis, was efficiently converted into uridine nucleotides and also incorporated into uracil and cytosine bases of nucleic acids. High activity of orotate phosphoribosyltransferase was observed in the extracts. A large proportion of radioactivity from [2‐¹⁴C]uracil was recovered as CO₂ and β‐ureidopropionate. Thus, a reductive pathway of uracil degradation is functional in these cells. Therefore, white spruce cells in culture demonstrate both the de novo and salvage pathways of purine and pyrimidine metabolism, as well as some degradation of the substrates into CO₂.     

The mechanism supplying acetyl-CoA for terpene biosynthesis in sweet potato with black rot: Incorporation of acetate-2-14C, pyruvate-3-14C and citrate-2,4-14C into ipomeamarone

Silica gel thin layer chromatography showed that acetate-2-¹⁴C,pyruvate-3-¹⁴C and citrate-2,4-¹⁴C were incorporated into ipomeamaronein sweet potato root tissues infected by Ceratocystis fimbriata.Rates of incorporation of ¹⁴C, from these 3 substances, intothe CHCl³-CH₃OH-soluble lipid fraction and ipomeamarone wereof the followingder: acetate > pyruvate > citrate ¹This paper constitutes Part 82 of the Phytopathological Chemistryof Sweet Potato with Black Rot and Injury (Received December 11, 1969; )     

Metabolism of ipomeamarone in sweet potato root slices before and after treatment with mercuric chloride or infection with Ceratocystis fimbriata

Furanoterpenes produced in sweet potato (Ipomoea batatas) root tissue in response to fungal infection or injury with mercuric chloride were metabolized to compounds negative to Ehrlich's reagent. The metabolism of ipomeamarone in sweet potato root tissue was enhanced in response to cut injury. The enhanced metabolism of ipomeamarone in the cut tissue was partly prevented by HgCl₂ treatment. The initial metabolite of ipomeamarone was identified as ipomeamaronol, 15-hydroxyipomeamarone. A time-course analysis of the metabolism of [¹⁴C]ipomeamarone by cut tissue indicated that ipomeamarone was converted to ipomeamaronol, which was further metabolized to unknown compounds.     

Acetyl CoA,a central intermediate of autotrophic CO2 fixation in Methanobacterium thermoautotrophicum

The pathway of autotrophic CO₂ fixation in Methanobacterium thermoautotrophicum has been investigated by long term labelling of the organism with isotopic acetate and pyruvate while exponentially growing on H₂ plus CO₂. Maximally 2% of the cell carbon were derived from exogeneous tracer, 98% were synthesized from CO₂. Since growth was obviously autotrophic the labelled compounds functioned as tracers of the cellular acetyl CoA and pyruvate pool during cell carbon synthesis from CO₂. M. thermoautotrophicum growing in presence of U-¹⁴C acetate incorporated ¹⁴C into cell compounds derived from acetyl CoA (N-acetyl groups) as well as into compounds derived from pyruvate (alanine), oxaloacetate (aspartate), -ketoglutarate (glutamate), hexosephosphates (galactosamine), and pentosephosphates (ribose). The specific radioactities of N-acetylgroups and of the three amino acids were identical. The hexosamine exhibited a two times higher specific radioactivity, and the pentose a 1.6 times higher specific radioactivity than e.g. alanine. M. thermoautotrophicum growing in presence of 3-¹⁴C pyruvate, however, did not incorporate ¹⁴C into cell compounds directly derived from acetyl CoA. Those compounds derived from pyruvate, dicarboxylic acids and hexosephosphates became labelled. The specific radioactivities of alanine, aspartate and glutamate were identical; the hexosamine had a specific radioactivity twice as high as e.g. alanine.The finding that pyruvate was not incorporated into compounds derived from acetyl CoA, whereas acetate was incorporated into derivatives of acetyl CoA and pyruvate in a 1:1 ratio demonstrates that pyruvate is synthesized by reductive carboxylation of acetyl CoA. The data further provide evidence that in this autotrophic CO₂ fixation pathway hexosephosphates and pentosephosphates are synthesized from CO₂ via acetyl CoA and pyruvate.     

Dark CO(2) Fixation and its Role in the Growth of Plant Tissue

Experiments were designed to determine the significance of dark CO₂ fixation in excised maize roots, carrot slices and excised tomato roots grown in tissue culture. Bicarbonate-¹⁴C was used to determine the pathway and amounts of CO₂ fixation, while leucine-¹⁴C was used to estimate protein synthesis in tissues aerated with various levels of CO₂.

Organic acids were labeled from bicarbonate-¹⁴C, with malate being the major labeled acid. Only glutamate and aspartate were labeled in the amino acid fraction and these 2 amino acids comprised over 90% of the ¹⁴C label in the ethanol-water insoluble residue.

Studies with leucine-¹⁴C as an indicator of protein synthesis in carrot slices and tomato roots showed that those tissues aerated with air incorporated 33% more leucine-¹⁴C into protein than those aerated with CO₂-free air. Growth of excised tomato roots aerated with air was 50% more than growth of tissue aerated with CO₂-free air. These studies are consistent with the suggestion that dark fixation of CO₂ is involved in the growth of plant tissues.

     

Utilization of Photosynthetically Fixed 14CO2 into Alkaloids in Relation to Primary Metabolites in Developing Leaves of Catharanthus roseus

Partitioning of current photosynthates towards primary metabolites and its simultaneous incorporation in leaf alkaloids was investigated in developing leaves of medicinally important Catharanthus roseus. Of the total ¹⁴CO₂ assimilated, the leaves at positions 1–6 fixed 8, 22, 25, 19, 13, and 8 %, respectively, and stem 3 %. Leaf fresh mass, chlorophyll content, and CO₂ exchange rate increased up to the third leaf. The total alkaloid content was highest in young actively growing leaves, which declined with age. Total ¹⁴C fixed and its content in ethanol soluble fraction increased up to the third leaf and then declined. The ¹⁴C content in primary metabolites such as sugars and organic acids was also highest in the 3^rd leaf. The utilization of ¹⁴C assimilates into alkaloids was maximum in youngest leaf which declined with leaf age. Hence the capacity to synthesize alkaloids was highest in young growing leaves and metabolites from photosynthetic pathway were most efficiently utilized and incorporated into alkaloid biosynthetic pathway by young growing leaves.     

Biosynthesis of 3-methylpentacosane in the cockroach Periplaneta americana.

The biosynthesis of 3-methylalkanes was investigated in the cockroach $\text{Periplaneta americana}$. Between 0.2 and 0.3 percent of the labelled acetate and propionate injected into the insect was incorporated into the cuticular hydrocarbons, compared to 0.01 percent for labelled isoleucine. Twenty-three ± four percent of the [2-¹⁴C]acetate, 42 ± 3 and 44 ± 4 percent of the [2-¹⁴C] and [3-¹⁴C]propionate, and 75 ± 5 percent of the [1-¹⁴C]propionate incorporated into the cuticular hydrocarbons was found in 3-methylpentacosane. These results indicate that propionate serves as the source of the branching methyl group, suggesting a pathway in which this precursor is incorporated during the penultimate step in 3-methylalkane biosynthesis in insects.     

Incorporation of Label from Acetate and Laurate into the Mannan of Leishmania donovani via the Glyoxylate Cycle

Leishmania donovani promastigotes in late-stationary phase incorporated label from [2-¹⁴C]acetate and [1-¹⁴C]laurate into the mannose residues of mannan, thus confirming the presence of a functional glyoxylate bypass in these parasitic protozoa. Isolated, washed calls also incorporated label from [2-¹⁴C]acetate and [1-¹⁴C]laurate into mannan during a 1-hr incubation in buffer. Glucose had no effect on label incorporation into mannan, but glutamate caused over a four-fold increase in incorporation from [2-¹⁴C]acetate and a 2.4-fold increase from [1-¹⁴C]laurate. Staurosporine, a protein kinase inhibitor that inhibits glutamate and alanine oxidation, did not inhibit label incorporation from [2-¹⁴C]acetate into mannan. Hyperosmolality caused about a 33% inhibition of label incorporation into mannan. These results show the glyoxylate cycle and/or the subsequent biosynthetic pathway from fructose-6-phosphate to mannan are subject to regulation.     

INORGANIC CARBON AND ACETATE ASSIMILATION IN BOTRYOCOCCUS BRAUNII(CHLOROPHYTA)1

Assimilation of ¹⁴CO₂ or ¹⁴C-acetate by the hydrocarbon producing alga Botryococcus braunii Kützing was investigated to determine the allocation of incorporated ¹⁴C among early metabolites of photosynthesis and secondary metabolites. When the cells were exposed to NaH¹⁴CO₃ for 10 sec, over 90% of incorporated ¹⁴C was detected in phosphoglycerate, suggesting that this alga assimilates inorganic carbon by the C-3 pathway. The distribution pattern of ¹⁴C in the number of metabolites revealed that organic acids, neutral sugars and amino acids were first labelled with ¹⁴C, and, after lag periods of a few minutes, lipids including hydrocarbon were increasingly labelled. Addition of 5 mM acetate to the culture medium did not affect the growth of this alga but enhanced cellular respiration. The incorporation of ¹⁴CO₂ into the lipid fraction was stimulated, but net photosynthesis was inhibited by the addition of acetate. ¹⁴C-acetate was incorporated into lipids at a very low rate in comparison with the rate of ¹⁴CO₂ incorporation.     

Cinnamic acid is a precursor of benzoic acids in cell cultures of Hypericum androsaemum L. but not in cell cultures of Centaurium erythraea RAFN

Benzoic acids are precursors of xanthone biosynthesis which has been studied in cell cultures of Hypericum androsaemum (Hypericaceae) and Centaurium erythraea (Gentianaceae). In both cell cultures, methyl jasmonate induces the intracellular accumulation of a new xanthone. Under these inductive conditions, feeding experiments were performed with [U-¹⁴C]L-phenylalanine, [7-¹⁴C]benzoic acid and [7-¹⁴C]3-hydroxybenzoic acid. All three precursors were efficiently incorporated into the elicited xanthone in H. androsaemum, whereas 3-hydroxybenzoic acid was the only precursor to be incorporated into xanthones in C. erythraea. In addition, an appreciable increase in phenylalanine ammonia-lyase activity occurred only in methyl-jasmonate-treated cell cultures of H. androsaemum. Benzoic acids thus appear to be formed by different pathways in the two cell cultures studied. In H. androsaemum, benzoic acid is derived from cinnamic acid by side-chain degradation. In C. erythraea 3-hydroxybenzoic acid appears to originate directly from the shikimate pathway. Received: 21 January 2000 / Accepted: 12 July 2000     

Decreased Synthesis of Glycosphingolipids in Cells Lacking Vimentin Intermediate Filaments

We are studying defects in glycosphingolipid synthesis in cells lacking vimentin intermediate filaments (vimentin−). Sugars can be incorporated into glycolipids whose ceramide is synthesized eitherde novo(pathway 1) or from sphingoid bases salvaged from hydrolysis of sphingolipids (pathway 2) and into glycolipids recycling from the endosomal pathway through the Golgi (pathway 3). Vimentin− embryonic fibroblasts, obtained from vimentin-knockout mice, incorporate less sugar into glycolipids than vimentin+ fibroblasts. Using two inhibitors of ceramide synthesis, β-chloroalanine and fumonisin B1, we found the major defect in synthesis to be in pathway 2 and not inde novosynthesis. We used two additional approaches to analyze the functions of pathways 2 and 3. First, we used exogenous glucosylthioceramide ([¹⁴C]C₈-Glc-S-Cer), a synthetic, nonhydrolyzable glycosphingolipid, as a precursor for synthesis of larger glycolipids. Vimentin− SW13 cells and embryonic fibroblasts glycosylated [¹⁴C]C₈-Glc-S-Cer less extensively than their vimentin+ counterparts. Second, we used chloroquine to inhibit the hydrolysis of sphingolipids in endosomes and lysosomes. Chloroquine markedly decreased the incorporation of sugars into glycolipids larger than glucosylceramide. The defect in glycolipid synthesis in vimentin− cells probably results from impaired intracellular transport of glycolipids and sphingoid bases between the endosomal/lysosomal pathway and the Golgi apparatus and endoplasmic reticulum. Intermediate filaments may accomplish this function by contributing to the organization of subcellular organelles and/or by binding proteins that participate in transport processes.     

The Role of β-Hydroxypropionate in Ethylene Biosynthesis

To search precursors of ethylene in banana fruits, ethylene formation from acetate-2-¹⁴C and fumarate-2,3-¹⁴C by banana slices was studied. Ethylene-¹⁴C formation from acetate-2-^l4C was reduced by the addition of malonate or β-hydroxypropionate, and it was enhanced in a sealed chamber in comparison with the case in an aeration chamber. No label of fumarate-2,3-¹⁴C was incorporated into ethylene.

From these facts it was suggested that acetate-2-¹⁴C was incorporated into ethylene via malonate and β-hydroxypropionate. Participation of fumarate in ethylene biosynthesis of banana fruits was ruled out. β-Hydroxypropionate was postulated as an effective precursor of ethylene formation from acetate-2-^l4C.     

Biosynthesis of vitamin B12

Radioactivity from [1-¹⁴C]riboflavin was incorporated into the 5,6-dimethylbenzimidazole moiety of Vitamin B₁₂ in the aerobes Bacillus megaterium, Nocardia rugosa and Streptomyces sp. as well as in the aerotolerant anaerobe Propionibacterium freudenreichii, but not in the anaerobe Eubacterium limosum.As recently published for E. limosum, also in the anaerobe Clostridium barkeri radioactivity from [1-¹⁴C]glycine and [2-¹⁴C]glycine was found in the 5,6-dimethylbenzimidazole moiety, but not in the corrin moiety. The addition of l-[methyl-¹⁴C]methionine to C. barkeri led to the labeling of the corrin moiety and the 5,6-dimethylbenzimidazole moiety, showing that the seven extra methyl groups in the corrin ring as well as the two methyl groups of the base part originate from this precursor.In Clostridium thermoaceticum, forming the vitamin B₁₂ analog 5-methoxybenzimidazolylcobamide, [1-¹⁴C]glycine and [2-¹⁴C]glycine were also incorporated into the 5-methoxybenzimidazole moiety, but not into the corrin ring.In E. limosum l-[U-¹⁴C]glutamate led to the labeling of the corrin ring of vitamin B₁₂, but not of its base moiety.There results together with data from the literature indicate that a common biosynthetic pathway might exist for the corrinoid biosynthesis in aerobic microorganisms, and in those aerotolerant anaerobes like the Propionibacteria, which form the 5,6-dimethylbenzimidazole moiety of vitamin B₁₂ only under aerobic conditions. They also show that this pathway differs from the pathway found in anaerobic bacteria.     

Incorporation of Shikimic Acid into Corynecins and Its Regulation

In the biosynthesis of corynecins by Corynebacterium hydrocarboclastus, it appeared that shikimic acid was one of the efficient precursors, where shikimic acid-U-¹⁴C was incorporated into corynecins in the yield of approximately 15%. Analyses of degradation products of labeled corynecins demonstrated that shikimic acid was incorporated specifically into aromatic ring of corynecins.

The incorporation of shikimic acid was inhibited by several aromatic amines such as p-aminophenylserinol-N-propionamide, although the uptake of shikimic acid was not affected, suggesting that biosynthesis of corynecins might be regulated by p-aminophenyl intermediates. Furthermore, p-ammophenylethylalcohol was found to be a potent inhibitor of biosynthesis of corynecins. In contrast, corynecins and other p-nitro-phenyl derivatives, aromatic amino acids and vitamins related to shikimic acid pathway did not inhibit the biosynthesis of corynecins from shikimic acid.     

Glycerolipid synthesis by homogenate and oil bodies from developing mustard (Sinapis alba L.) seed

[1-¹⁴C]Oleic acid and [14-¹⁴C]erucic acid were converted to their acyl-CoA derivatives and incorporated into acyl lipids by a homogenate from developing mustard (Sinapis alba L.) seed and oil bodies, as well as supernatant isolated by centrifugation at 20000 g. In both homogenate and oil bodies, the oleoyl moieties from exogenous [1-¹⁴C]oleoyl-CoA were most extensively incorporated into phosphatidic acids, but very little into phosphatidylcholines. The pattern of labelling of acyl lipids by oleoyl versus erucoyl moieties from either of the corresponding fatty acids, added individually or as a mixed substrate, indicates that oleoyl-CoA directly acylates sn-glycerol-3-phosphate to yield lysophosphatidic acids and phosphatidic acids that are subsequently converted to mono- and diacylglycerols. In contrast, erucoyl-CoA predominantly acylates preformed mono-and diacylglycerols containing oleoyl moieties to yield triacylglycerols containing erucoyl moieties.     

Obligately phototrophic Chloroflexus: primary production in anaerobic hot spring microbial mats

Microbial mats which lack cyanobacteria occur at 50° to 65° C in the sulfide-containing Mammoth Springs of Yellowstone National Park. The principal organisms within these mats are filamentous bacteria which resemble Chloroflexus aurantiacus. The incorporation of [¹⁴C]-HCO ₃ ^- into mat material depended upon both light and sulfide, and was not inhibited when complete natural light was replaced with far-red and infra-red radiation. [¹⁴C]-acetate was incorporated in a light-dependent reaction which was stimulated by, but did not require, sulfide. In situ experiments with microelectrodes demonstrated net sulfide uptake by the mat in the light, and net sulfide production by the mat in the dark, suggesting the operation of a sulfur cycle.Filamentous phototrophic bacteria isolated from the mat were incapable of sustained growth in the presence of O₂.Simultaneous exposure of cultures to light and O₂ caused degradation of bacteriochlorophyll c. The stimulation of light-dependent [¹⁴C]-HCO ₃ ^- -uptake by sulfide was more pronounced in these isolates than in strains of Chloroflexus aurantiacus.