The Biosynthesis of (+)-Tartaric Acid in Pelargonium crispum

Ochromoaas danica Pringsheim, a freshwater chrysomonad, converts D-glucose into L-ascorbkc acid over a metabolic pathway that 'inverts' the carbon chain of the sugar. In this respect, L-ascorbic acid formation resembles that found in ascorbic acid-synthesizing animals. It differs from this process in that D-galacturonate and L-galactono-1,4-Iactone, rather than D-gucuronate and L-guloo-1,4-lactone, enhance production of ascorbic acid and repress the incorporation of 14C from D-l1-14ClgIucose into ascorbic acid.Two mutually exclusive, biosynthetic pathways of AA3 formation are found in eukaryotic organisms. In both, D-glucose is implicated as the six-carbon precursor on the basis of studies with radioactive specifically labeled sugars. Both pathways conserve the six-carbon chain. One 'inverts' the sequence of carbons, as referred to the parent sugar by reduction of C-1 and oxidation of C-5 (or C4) and C-6 with conservation of the chirality of C-2 and C-3. AA-Synthesizing animals such as the rat utilize this process according to the following scheme:lactone -. L-Ascorbate. The other pathway requires that C-I and C-2 (or C-3) be oxidized while an epimerization reverses the configuration of C-5. Higher plants utilize this latter process, which conserves the hydroxymethyl function of C-6 of hexose (15) 4-lactone (4, 6, 9, 19). Inasmuch as radiolabeling studies exclude these compounds as intermediates in the overall pathway from D-glucose to AA, these synthetic events appear to be dependent upon the exogenous source or, at best, salvage mechanisms (7 and L-galactono-1,4-lactone were intermediates. D-Glucurono-3,6-lactone and L-gulono-1,4-lactone also effected AA synthesis, and these compounds were included in their scheme as a minor alternate route that differed from the animal pathway in one respect, reduction of D-glucurono-3,6-lactone to L-gulono-1,4-lactone rather than reduction of D-glucuronate to L-gulonate (23).Ochromonas danica Pringsheim, a unicellular chrysophycean alga with phagotrophic, heterotrophic, photoheterotrophic, and photoautotrophic properties, provides an exceptional opportunity to explore the phylogenetic connection between animal and plant as it pertains to AA biosynthesis. 0. danica synthesizes and secretes into the medium a number of vitamins, including AA, when grown in an artificial medium (2). Advantage has been taken of this observation to explore the conversion of specifically labeled D-glucose into AA and to test the effect of possible intermediates in this process. MATERIALS AND METHODSChemicals. L-[l-14C]Ascorbic acid and specifically labeled forms of D-glucose were purchased from New England Nuclear Corp. Chemicals, including those used to prepare culture media, were reagent grade.Algal Culture. 0. danica Pringsheim was obtained from the Culture Collection of Algae at the University of Texas at Austin. Cells were dark-grown at 25°C in chemically defined medium containing 1% D-glucose (1). Inoculum (10 ml, approx. 108 cells) was added to 100 ml of medium. After 14 d, the enti...

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l-Ascorbic Acid Biosynthesis in Ochromonas danica

Helsper¹,

Kagan²,

Hilby³

et al. 1982

“…Our interest in this problem stemmed from studies on tartaric acid biosynthesis in Pelargonwm crispum (L.) L'Her. (lemon geranium) (2). In KOH that was suspended in the metabolic flask.…”

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confidence: 99%

Further Studies on Oxalic Acid Biosynthesis in Oxalate-accumulating Plants

Nuss

Loewus²

1978

The present study extends this survey on the metabolism of [ -'4CJAA to other oxalate-accumulating plants and to some which contain low amounts of oxalate. It also presents evidence that C l+C2 of AA is the fragment utilized for oxalic acid biosynthesis. Batches of seedlings for use in labeling studies were grown on moist filter paper under sterile conditions. Chemcals. Labeled AA were purchased from New England MATERIALS AND METHODS

“…This latter observation prompted a careful study of the isomeric form of labeled TA recovered from Pelargonium crispum L. after feeding L-ascorbic acid-6-4C (28) and from Vitis labrusca L. and Parthenocissus inserta after feeding L-AA-1-"4C (29). Data feeding D-glucose-1-`C or -6-'4C were also gathered (12 L-AA-6-'4C (28) and from Vitis labrusca berries and Parthenocissus inserta leaves which were labeled with L-AA-1-"4C (29) were used in this study.…”

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confidence: 99%

“…Only the (+) form has received much attention as regards its metabolic origin. Literature pertinent to biosynthesis of (+) TA is summarized in recent publications (19,(27)(28)(29). These biosynthetic studies employed labeled precursors and, in most instances, recovered TA from solution as its poorly soluble KH salt.…”

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confidence: 99%

“…Data feeding D-glucose-1-`C or -6-'4C were also gathered (12 L-AA-6-'4C (28) and from Vitis labrusca berries and Parthenocissus inserta leaves which were labeled with L-AA-1-"4C (29) were used in this study. Samples from P. crispum apices which were labeled for 72 hr with D-glucose-1-"C or -6-"'C by the same procedure used in AA experiments (12) were also studied.…”

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confidence: 99%

See 1 more Smart Citation

Stereoisomeric Characterization of Tartaric Acid Produced during l-Ascorbic Acid Metabolism in Plants

Wagner¹,

Yang²,

Loewus³

1975

Self Cite

Labeled tartaric acids from Pelargoniunt crispum apices which had been fed L-ascorbic acid-6-"C and Vitis labrusca and Parthenocissus inserta tissues which had been fed L-ascorbic acid-l-"4C were examined by chemical means to determine chiral configuration. In each instance, label was associated with (+)-tartaric acid.Similar experiments with labeled tartaric acid from P.crispuom which had been labeled with D-glucose-l-"C or -6-'4C led to the same result. No evidence was obtained for formation of labeled meso-tartaric acid in experiments described above.The recent suggestion of H. Ruffner and D. Rast (Z. Pflanzenphysiol. 73: 45-55, 1974) that conversion of L-ascorbic acid to tartaric acid in plants is a nonenzymatic process is re-examined in the light of present findings.All three stereoisomeric forms of tartaric acid have been found in higher plants; (+) TA' in numerous plant families (4) notably the Vitaceae and Geraniaceae (23, 24); (-) TA in Bauhinia reticulata (18) and as esters of caffeic acid in Cichorium intybus L., C. endivia L. and Lactuca sativa L. (6,21,22,30); and meso-TA as an ester of p-coumaric acid in spinach leaves and isolated spinach chloroplasts (16,25) or as an excreted product (5). Only the (+) form has received much attention as regards its metabolic origin. Literature pertinent to biosynthesis of (+) TA is summarized in recent publications (19,(27)(28)(29). These biosynthetic studies employed labeled precursors and, in most instances, recovered TA from solution as its poorly soluble KH salt. In one study (13,14), formation of (+) TA from glycolate-1-"C in Pelargonium zonale L. was accompanied by (-) TA and/or meso TA.This latter observation prompted a careful study of the isomeric form of labeled TA recovered from Pelargonium crispum L. after feeding L-ascorbic acid-6-4C (28) L-AA-6-'4C (28) and from Vitis labrusca berries and Parthenocissus inserta leaves which were labeled with L-AA-1-"4C (29) were used in this study. Samples from P. crispum apices which were labeled for 72 hr with D-glucose-1-"C or -6-"'C by the same procedure used in AA experiments (12) were also studied.Separation of Diastereomeric Forms of TA. Ion exchange chromatography with Dowex 1 (formate) resin and a 0 to 4 M formic acid gradient readily separated meso-TA from the chiral forms. With the two step gradient procedure described earlier (28), meso-TA appeared in the second step of the gradient in the volume eluted between 60 to 110 ml and chiral TA between 100 to 160 ml. Further purification by paper chromatography (28) and GLC (27) was used to identify TA.Resolution of Labeled TA. Samples used for this study had been recovered from plant extracts by ion exchange chromatography using (+) TA as carrier. Chiral TA fractions were combined and adjusted to pH 3.5 with KOH to recover the KH salt of TA. A weighed aliquot containing 0.05 to 0.1 1,Ci was converted to free TA and combined with an equimolar amount of (-) TA to make a racemic mixture. Following further dilution with unlabeled racemic TA, the mixture w...