Metabolism of [2-3H]- and [6-3H]-nicotinic acid in intact Nicotiana tabacum plants

Earlier observations of Dawson on the relative incorporation of [2-³H]- and [6-³H]-nicotinic acid into nicotine have been confirmed in intact Nicotiana tabacum plants. All the tritium in the nicotine derived from [2-³H]-nicotinic acid was located at C-2 of the pyridine ring. However the radioactive nicotine derived from [6-³H]-nicotinic acid was not labelled specifically at C-6 with tritium. By carrying out feeding experiments with [6-¹⁴-C, 2-³H]- and [6-¹⁴C, ³H]-nicotinic acids, it was established that there was very little loss of tritium from C-2 and C-6 of nicotinic acid during 5 days of metabolism in the tobacco plant.     

Production of a co-polyester of 3-hydroxybutyric acid and 3-hydroxyvaleric acid from succinic acid by Rhodococcus ruber: biosynthetic considerations

The biosynthesis of the 3-hydroxyvalerate (3HV) monomer of polyhydroxyalkanoate by Rhodococcus ruber from succinic acid was investigated using nuclear magnetic resonance analysis. Polymer produced from [2,3-¹³C]- and [1,4-¹³C]succinate showed that the C-1-C-2 and C-4-C-5 fragments of 3HV were derived from carbons 2 and 3 of succinate, essentially without bond cleavage, and carbon 3 of 3HV was derived from a carboxyl carbon of succinate. Using [1,2-¹³C]succinate it was demonstrated that the C-1-C-2 bond of succinate was cleaved during polymer biosynthesis. Methylmalonyl-coenzyme A (CoA) mutase activity was detected in cell-free extracts of R. ruber by enzyme assay and HPLC analysis of reaction products. A pathway, involving the known methylmalonyl-CoA pathway for propionate formation in Propionibacteria, followed by the established pathway for PHA biosynthesis from propionyl-CoA and acetyl-CoA, is proposed for the biosynthesis of 3HV from succinate by R. ruber. Correspondence to: A. J. Anderson     

Different biosynthetic pathways of the pyrimidine moiety of thiamin in procaryotes and eucaryotes

[¹⁴C]Formate is incorporated into the C-2 of the pyrimidine moiety of thiamin by Escherichia coli and Salmonella typhimurium. In Saccharomyces cerevisiae, it is incorporated into C-4. Radioactive carbons of [1-¹⁴C]glycine and [2-¹⁴C]glycine are incorporated by S. typhimurium into the C-4 and C-6 of the pyrimidine, respectively, but not by S. cerevisiae. These facts suggest that procaryotes and eucaryotes have different biosynthetic pathways for pyrimidine. In this study, the procaryotes tested incorporated [¹⁴C]formate into the C-2 and the eucaryotes incorporated it into the C-4 of the pyrimidine.     

Biosynthesis of chloramphenicol. Studies on the origin of the dichloroacetyl moiety

Chloramphenicol produced by cultures of Streptomyces species 3022a supplemented with sodium [1,2-13C]acetate was labelled with 13C exclusively in the dichloromethine (2.6 +/- 0.1%) and carbonyl (0.59 +/- 0.05% carbon atoms. Satellite signals from 13C-13C coupling between covalently bonded 13C-enriched carbon atoms were too intense to be attributed to random combination of labelled atoms at the average enrichments measured, but their intensity relative to those of the signals for uncoupled 13C atoms indicated that most of the precursor had been incorporated after 13C-13C bond fission. Since [2,3-13c]succinic acid enriched only the carbonyl carbon atom of chloramphenicol, these results suggest that neither acetate nor a Krebs cycle intermediate is a direct precursor of the dichloroacetyl group. Cultures supplemented with [2-3h]-or [2h2]-dichloroacetic acid incorporated negligible amounts of isotope into the antibiotic; on this evidence, the free acid is not an intermediate in chloramphenicol biosynthesis and the acylation step may precede chlorination.     

Comparative study of in vivo stigmasterol biosynthesis in Nicotiana tabacum and Hordeum vulgare

Six-day-old tobacco (Nicotiana tabacum) and barley (Hordeum vulgare) seedlings rapidly incorporated and metabolized exogenously supplied [4-¹⁴C]sitosterol but neither plant was able to convert it into stigmasterol. However, a sterol metabolite was isolated from both species and the acetate derivative was slightly more polar, on AgNO₃—silica gel TLC, than stigmasteryl acetate. A similar metabolite was also obtained with [4-¹⁴C]cholesterol, indicating a general metabolic reaction of plants to exogenous sterols. Both species incorporated [2-¹⁴C]mevalonic acid into sitosterol and stigmasterol. We suggest that in vascular plants, whether monocotyledons or dicotyledons, the pathway of stigmasterol biosynthesis is not via sitosterol but through a common precursor which is derived from mevalonic acid.     

A method for the degradation of radioactive nicotinic acid

A chemical degradation scheme is reported, which permits the measurement of the radioactivity of each carbon atom of nicotinic acid. Nicotinic acid is decarboxylated by heating with copper chromite to give carbon dioxide (C-7) and pyridine. The pyridine is converted into 4-nitropyridine 1-oxide, which is heated with aqueous calcium hypobromite to give tribromonitromethane. Combustion of the latter gives carbon dioxide derived from C-4 of the nicotinic acid. Nicotinic acid is also reduced to nipecotic acid, which is oxidized to succinic acid by acidic potassium permanganate. Stepwise degradation of the succinic acid by standard procedures gives two samples of carbon dioxide, which correspond to C-3, C-6 and C-4, C-5 of the nicotinic acid. Benzoylation of the nipecotic acid, followed by oxidation with permanganate at pH7, gives 5-amino-4-carboxyvaleric acid; this is converted into 2-methyleneglutaric acid by the action of nitrous acid. Hydrogenation of the 2-methyleneglutaric acid over rhodium in methanol gives 2-methylglutaric acid, which is oxidized with dilute chromic acid to acetic acid. Stepwise degradation of the acetic acid by standard procedures gives two samples of carbon dioxide, which correspond to C-2 and C-3 of the nicotinic acid. Thus the radioactivities of C-2, C-3, C-4 and C-7 are determined directly and those of C-5 and C-6 by difference. The method was shown to be isotopically valid for [2,3,7-¹⁴C]-, [4,6-¹⁴C₂]- and [5-¹⁴C]-nicotinic acid.     

The stereochemistry of biosynthesis of decaprenoxanthin in a cell-free system

Intact cells of Flavobacterium dehydrogenans grown on glucose or acetate did not incorporate mevalonic acid-[¹⁴C]. After treatment with lysozyme the protoplasts were lysed by sonication in a dilute medium containing mevalonic acid-[¹⁴C] and the cell-free system produced incorporated label into uncyclized C₄₀, monocyclic C₄₅ and bicyclic C₅₀ carotenoids of which decaprenoxanthin was the most abundant.With mevalonate-[2-¹⁴C,4R-4-³H₁] the ¹⁴C:³H ratios of the carotenoids showed that the hydrogen atoms at C-2 and C-6 of the ring and that at C-3 of the 1-hydroxy, 2-methyl but-2-ene-4-yl residues of decaprenoxanthin were derived from the 4-pro-R hydrogen atom of mevalonic acid.Mevalonate-[2-¹⁴C,2R-2-³H₁] and mevalonate-[2-¹⁴C,2S-2-³H₁] gave ratios which showed that the C-4 hydrogen atoms of decaprenoxanthin were derived from the 2-pro-S hydrogen atom of mevalonic acid.     

Arginine metabolism in developing soybean cotyledons : I. Relationship to nitrogen nutrition

The free and protein amino acid composition of Glycine max (L.) Merrill cotyledons was determined for the entire developmental period using high performance liquid chromatography. Arginine constituted 18% of the total protein nitrogen throughout development, and there was a linear arginine nitrogen accumulation rate of 1212 nanomoles per cotyledon per day between 16 and 58 days after anthesis. Arginine and asparagine were major constituents of the free amino acid pool, constituting 14 to 62% and 2 to 41% of the total free amino acid nitrogen, respectively. The urea cycle intermediates, citrulline, ornithine, and argininosuccinate were also detected in the free pool. A comparison of the amino acid composition of cotyledonary protein and of seedcoat exudate suggested that 72% of the cotyledon's arginine requirement is satisfied by in situ biosynthesis, and that 20% of the transformed nitrogen is incorporated into arginine. Also, [1-¹⁴C]glutamate and [U-¹⁴C]glutamine were fed to excised cotyledons. After 4 hours, ¹⁴C was incorporated into protein and released as ¹⁴CO₂, but none was incorporated into the C-1 and C-6 positions of free and protein arginine, determined using arginine-specific enzyme-linked assays. It is not currently known whether arginine biosynthesis in the cotyledon involves glutamate delivered from the mother plant or glutamate derived in situ.     

Biosynthesis of artemisia ketone in higher plants

MVA-[2-¹⁴C], IPP-[4-¹⁴C] and DMAPP-[4-¹⁴C] were incorporated (optimum 0.04%–0.8 %) into artemisia ketone by Artemisia annua in a position-specific manner so that the C-5 moiety not containing the carbonyl group was preferentially (87–95 %) labelled. IPP and DMAPP, but not MVA, were similarly utilised in Santolina chamaecyparissus. Feeding of geraniol-[2-¹⁴C] to A.annua resulted in artemisia ketone being labelled in a position indicating extensive degradation of the precursor. ¹⁴C-labelled cis and trans-chrysanthemyl alcohols and chrysanthemates or DMVC were negligibly (< 5 × 10^?4 %) incorporated into artemisia ketone in both species over a range of feeding conditions. (+)-trans-Chrysanthemyl alcohol-[Me¹⁴C] was an effective (ca 2 % incorporation) precursor of the terpenoid part of pyrethrins I and II in flowers of Chrysanthemum cinerariaefolium but ¹⁴C-labelled artemisyl alcohol (3, 3, 6-trimethylheptan-1, 5-dien-4-ol) or (±)-cis-chrysanthemyl alcohol were not detectably incorporated. Although some of the negligible incorporations are probably attributable to compartmentation effects preventing access of precursors to biosynthetic sites, the experiments indicate some limitation of the previously proposed pathways of biogenesis of artemisia ketone and related irregular monoterpenes.     

Conversion of 5-(1,2-epoxy-2,6,6-trimethylcyclohexyl) -3-methylpenta-cis-2-trans-4-dienoic acid into abscisic acid in plants

(±)-5-(1,2-Epoxy-2,6,6-trimethylcyclohexyl) -3-methyl[2-¹⁴C]penta-cis-2-trans-4-dienoic acid is converted into abscisic acid by tomato fruit in 1.8% yield (or 3.6% of one enantiomer if only one is utilized) and 15% of the abscisic acid is derived from the precursor. The 2-trans-isomer is not converted. The amounts of [2-³H]mevalonate incorporated into abscisic acid have shown that the 40-times higher concentration of (+)-abscisic acid in wilted wheat leaves in comparison with unwilted ones reported by Wright & Hiron (1969) arises by synthesis. The conversion of (±)-5-(1,2-epoxy-2,6,6-trimethylcyclohexyl) -3-methyl-[2-¹⁴C]penta-cis-2-trans-4-dienoic acid into abscisic acid by wheat leaves is also affected in the same way by wilting and it is concluded from this that the epoxide or a closely related compound derived from it is on the biosynthetic pathway leading to abscisic acid. The oxygen of the epoxy group was shown, by ¹⁸O-labelling, to become the oxygen of the tertiary hydroxyl group of abscisic acid.     

Metabolism of alkyl glyceryl ethers and their noninvolvement in alkane biosynthesis in plants

[1-¹⁴C]Octadecyl glyceryl ether did not label alkanes in the leaves of Brassica oleracea and Pisum sativum while [1-¹⁴C]octadecanol and [1-¹⁴C]octadecanoic acid readily labeled the alkanes. About 40% of the exogenous-labeled glyceryl ether was incorporated intact into choline phosphatide while 10–20% was converted into fatty acids and alcohols. [1-¹⁴C]octadecanol was not converted into alkyl glyceryl ether, but it was oxidized to the corresponding acid and then incorporated into alkanes. These results show that alkyl ether is not an intermediate in alkane biosynthesis. When [1-¹⁴C-1-³H]-octadecanol was fed to the leaves of B. oleracea and P. sativum, only the ¹⁴C and no ³H was incorporated into alkanes, ketones, and secondary alcohols. These results show that fatty alcohols are first oxidized to the acid before being incorporated into alkanes, ruling out fatty alcohol, alkyl ether, and alk-1-enyl ether as intermediates in alkane biosynthesis. The exogenous alcohols were also readily esterified into wax esters in both tissues.     

Biosynthesis of the 2-arylbenzofuran phytoalexin vignafuran in Vigna unguiculata

Feeding experiments using l-phenylalanine-[U-¹⁴C], dl-phenylalanine-[1-¹⁴C] and -[2-¹⁴C] together with degradative studies have been used to investigate the biosynthesis of the 2-arylbenzofuran phytoalexin vignafuran in UV-treated seedlings of cowpea (Vigna unguiculata). During the biosynthetic process, C-3 of phenylalanine appears to be lost, and the resulting labelling pattern is consistent with vignafuran being derived from an isoflavonoid precursor, but the phenylalanine-derived aromatic ring becomes the 2-aryl substituent and not part of the benzofuran system. A previously proposed pathway to 2-arylbenzofurans by loss of C-6 from a coumestan is thus excluded. Alternative routes are suggested.     

1,2 Hydrogen-shifts in the biosynthesis of the thujane skeleton

Degradation of (+)-isothujone (trans-thujan-3-one) biosynthesized in Tanacetum vulgare from (3RS)-mevalonic acid (MVA)-[2-¹⁴C, 2-³H₂] showed that one hydrogen from C-2 of the precursor was specifically incorporated at C-4 of product whereas the other was lost. Feeding of α-terpineol-[9-¹⁴C, 4-³H₁, 10-³H₃] (p-menth-1-en-8-ol) yielded isothujone with the same isotope ratios as in precursor. These results indicate 1,2 hydrogen-shifts at two locations in the construction of the the thujane skeleton from α-terpineol or its biogenetic equivalent, and are consistent with a mechanism involving direct cyclization of the latter to a product that by-passes the formation of the biogenetic equivalent of terpinen-4-ol (p-menth-1-en-4-ol) as an intermediate. (3R)-MVA-[¹⁴C, ³H] was more effectively incorporated (up to 1.5 %) into (+)-isothujone in vivo during autumn or winter than in summer (up to 0.02%).     

6-Ethyl-5-hydroxy-2,7-dimethoxy-1,4-naphthoquinone from Hendersonula toruloidea: A biosynthetic study using 13C labels detected by nuclear magnetic resonance and 14C tracers

Production of 6-ethyl-5-hydroxy-2,7-dimethoxy-1,4-naphthoquinone was obtained by growth of Hendersonula toruloidea on Czapek-Dox broth supplemented with malt extract. Stationary cultures were grown at 28°C for 21–22 days yielding about 6 mg of metabolite per 700 ml of culture fluid. The best incorporations of isotopic tracers were obtained by addition at the 20th day of growth, followed by harvest 24–48 hr later. With [2-¹⁴C]acetate, incorporation values were in the range of 0.1–0.3% with dilution values from 2000 to 5900. With [1-¹⁴C]propionate, incorporations were much lower (0.04%) and dilutions much higher (120,000). Activity from [${}^{14}\text{CH}{}_3$]methionine was incorporated only into the OCH₃ groups (incorporation values, 0.5–0.7%). Nuclear magnetic resonance studies confirmed that propionate was not a precursor. Using [1,2-¹³C]acetate, substantial enrichments were obtained at all carbon atoms except those of the OCH₃ groups. The following pairs of carbon atoms were shown to be derived from acetate units: C-1 + 2, C-3 + 4, C-5 + 10, C-6 + 7, C-8 + 9, C-11 + 12. The biosynthetic pathway is clearly that of acetate plus polymalonate. Experiments with [2-¹³C²H₃]acetate suggested that the “starter” acetate unit was located at positions C-12 + 11.     

Incorporation of shikimate and 4-(2′-carboxyphenyl)-4-oxobutyrate into phylloquinone

The patterns of incorporation of d-[G-¹⁴C]shikimate and variously labelled ¹⁴C-4-(2′-carboxy-phenyl)-4-oxobutyrate into the naphthoquinone nucleus of phylloquinone by maize shoots have been investigated. The results show that (a) the alicyclic ring and C-7 of shikimate give rise to Ring A and either C-1 or C-4, and (b) the phenyl ring, 2′-carboxy and C-4, and C-2 and -3 of 4-(2′-carboxyphenyl)-4-oxobutyrate give rise to Ring A, C-1 and -4 and C-2 and -3. Radioactivity from α-[1-¹⁴C]naphthol, 1,4-[1,4-¹⁴C]naphthoquinone and [Me-¹⁴C]menadione is not incorporated into phylloquinone to any significant extent.     

Biosynthesis of riboflavine by a purine-requiring mutant strain of Escherichia coli

1. Riboflavine biosynthesis occurs in non-proliferating cultures of a purine-requiring strain of Escherichia coli (ATCC no. 13863). 2. No significant incorporation of radioactivity from [1-¹⁴C]glycine into either C-4a and C-9a of riboflavine or into nucleic acid purines is detected under the above conditions; appreciable incorporation of label into 5-aminoimidazole-4-carboxamide occurs. However, the label of [6-¹⁴C]guanine is incorporated significantly into C-4 of riboflavine and into nucleic acid adenine and guanine; the specific radioactivity of the riboflavine is approximately twice that of either adenine or guanine of nucleic acid. 3. These results show that a purine derivative is an obligatory intermediate in riboflavine biogenesis.     

The involvement of sucrose,glucose and other metabolites in the synthesis of triterpenes and dopa in the laticifers of Euphorbia lathyris

A quantitative triterpene analysis was made of latex stem tissue of Euphorbia lathyris. Young plants seedlings of E. lathyris were incubated with various labelled precursors. Incorporation into triterpenes was obtained from [2-¹⁴C]mevalonic acid, [1-¹⁴C]acetate, [3-¹⁴C]pyruvate, [U-¹⁴C]sucrose, [U-¹⁴C]glucose, [U-¹⁴C]xylose, [U-¹⁴C]glyoxylate, [2,3-¹⁴C]succinic acid, [1-¹⁴C]glycerol [U-¹⁴C]serine. Both sugars tyrosine appeared to be effective precursors in DOPA synthesis inside the laticifers. Exogenously supplied mevalonic acid was only involved in triterpene synthesis outside the laticifers. GC-RC of triterpenes synthesized from [U-¹⁴C]glucose revealed the origin of these compounds in the latex. The labelled triterpenes obtained after incorporation of the other mentioned labelled precursors were only partly synthesized in the laticifers. For quantitative data on latex triterpene synthesis seedlings were incubated with [U-¹⁴C]sucrose, [U-¹⁴C]glucose, [U-¹⁴C]xylose [1-¹⁴C]acetate in the presence of increasing amounts of unlabelled substrate. From the amount of ¹⁴C incorporated into the triterpenes the amount of substrate directly involved in triterpene synthesis was calculated, as was the absolute triterpene yield. Sucrose showed the highest triterpene yield, equivalent to the daily increase of the triterpene content of growing seedlings. The possible significance of the other precursors in triterpene synthesis in the laticifers is discussed.     

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 azetidine-2-carboxylic acid in Convallaria majalis

Convallaria majalis plants were fed dl-methionine-[1-¹⁴C]. [1-¹⁴C, 4-³H], and [1-¹⁴C, 2-³H], S-adenosyl-l-methionine-[1-¹⁴C], and dl-homoserine-[1-¹⁴C], resulting in the formation of labeled azetidine-2-carboxylic acid (A-2-C). The complete retention of tritium relative to carbon-14 in the feeding experiment involving methionine-[1-¹⁴C, 4-³H] indicates that aspartic acid or aspartic-β-semialdehyde are not intermediates between methionine and A-2-C. However, since the A-2-C derived from methionine-[1-¹⁴C, 2-³H] had lost 95% of the tritium relative to the C-14, it is not considered that methionine or its S-adenosyl derivative are the immediate precursors of A-2-C. Our data and that of others is consistent with the intermediate formation of γ-amino-α-ketobutyric acid which on cyclization yields 1-azetine-2-carboxylic acid, A-2-C then being formed on reduction.     

Gut tract microorganisms supply the precursors for methyl-branched hydrocarbon biosynthesis in the termite,Zootermopsis nevadensis

In vivo and in vitro experiments were performed to examine the role of succinate and other potential precursors of the methylmalonyl-CoA used for methyl-branched hydrocarbon biosynthesis in the termite Zootermopsis nevadensis. The in vivo incorporation of [1,4-¹⁴C]succinate and [2,3-¹⁴C]succinate into hydrocarbon confirmed that succinate is a direct precursor to the methyl branch unit. The other likely precursors, the branched chain amino acids valine and isoleucine, were not efficiently incorporated into hydrocarbon. Carbon-13 NMR showed that one of the labeled carbons of [1,4-¹³C]succinate labeled position 6 of 5-methylalkanes and positions 6 and 18 of 5,17-dimethylalkanes, indicating that succinate, as a methylmalonyl-CoA unit, was incorporated as the third unit to form 5-methylheneicosane and as both the third and ninth units to form 5,17-dimethylheneicosane. Analysis of organic acids after the in vivo metabolism of [2,3-¹⁴C]succinate showed that succinate was converted to propionate and methylmalonate. Labeled succinate injected into the hemolymph was readily taken up by the gut tract. Isolated gut tissue efficiently converted succinate to acetate and propionate, both of which were released into the incubation media. Mitochondria from termite tissue (minus gut tract) converted succinate to methylmalonate and propionate only in the presence of malonic acid, an inhibitor of succinate dehydrogenase. The results of these studies show that while termite mitochondria are able to convert succinate to propionate and methylmalonate, most of the propionate used for methyl-branched hydrocarbon biosynthesis is produced by gut tract microorganisms. The propionate is then presumably transported through the hemolymph to epidermal cells for use in methyl-branched hydrocarbon biosynthesis.