ascorbic-acid and galactonic-acid

Ascorbate is an abundant and indispensable redox compound in plants. Genetic and biochemical studies have established the d-mannose/l-galactose (d-Man/l-Gal) pathway as the predominant ascorbate biosynthetic pathway in streptophytes, while the d-galacturonate (d-GalUA) pathway is found in prasinophytes and euglenoids. Based on the presence of the complete set of genes encoding enzymes involved in the d-Man/l-Gal pathway and an orthologous gene encoding aldonolactonase (ALase) - a key enzyme for the d-GalUA pathway - Physcomitrium patens may possess both pathways. Here, we have characterized the moss ALase as a functional lactonase and evaluated the ascorbate biosynthesis capability of the two pathways using knockout mutants. Physcomitrium patens expresses two ALase paralogs, namely PpALase1 and PpALase2. Kinetic analyses with recombinant enzymes indicated that PpALase1 is a functional enzyme catalyzing the conversion of l-galactonic acid to the final precursor l-galactono-1,4-lactone and that it also reacts with dehydroascorbate as a substrate. Interestingly, mutants lacking PpALase1 (Δal1) showed 1.2-fold higher total ascorbate content than the wild type, and their dehydroascorbate content was increased by 50% compared with that of the wild type. In contrast, the total ascorbate content of mutants lacking PpVTC2-1 (Δvtc2-1) or PpVTC2-2 (Δvtc2-2), which encode the rate-limiting enzyme GDP-l-Gal phosphorylase in the d-Man/l-Gal pathway, was markedly decreased to 46 and 17%, respectively, compared with that of the wild type. Taken together, the dominant ascorbate biosynthetic pathway in P. patens is the d-Man/l-Gal pathway, not the d-GalUA pathway, and PpALase1 may play a significant role in ascorbate metabolism by facilitating dehydroascorbate degradation rather than ascorbate biosynthesis.

Topics: Ascorbic Acid; Bryopsida; Carboxylic Ester Hydrolases; Galactose; Gene Expression Regulation, Plant; Gene Knockout Techniques; Genome, Plant; Kinetics; Light; Mannose; Metabolic Networks and Pathways; Mutation; Phenotype; Plant Proteins; Sugar Acids

We have previously proposed that Euglena gracilis possesses a pathway for the production of ascorbate (AsA) through d-galacturonate/L-galactonate as representative intermediates ( Shigeoka, S., Nakano, Y., and Kitaoka, S. (1979) J. Nutr. Sci. Vitaminol. 25, 299-307 ). However, genetic evidence proving that the pathway exists has not been obtained yet. We report here the identification of a gene encoding aldonolactonase, which catalyzes a penultimate step of the biosynthesis of AsA in Euglena. By a BLAST search, we identified one candidate for the enzyme having significant sequence identity with rat gluconolactonase, a key enzyme for the production of AsA via d-glucuronate in animals. The purified recombinant aldonolactonase expressed in Escherichia coli catalyzed the reversible reaction of L-galactonate and L-galactono-1,4-lactone with zinc ion as a cofactor. The apparent K(m) values for L-galactonate and L-galactono-1,4-lactone were 1.55 +/- 0.3 and 1.67 +/- 0.39 mm, respectively. The cell growth of Euglena was arrested by silencing the expression of aldonolactonase through RNA interference and then restored to the normal state by supplementation with L-galactono-1,4-lactone. Euglena cells accumulated more AsA on supplementation with d-galacturonate than d-glucuronate. The present results indicate that aldonolactonase is significant for the biosynthesis of AsA in Euglena cells, which predominantly utilize the pathwayviad-galacturonate/L-galactonate. The identification of aldonolactonase provides the first insight into the biosynthesis of AsA via uronic acids as the intermediate in photosynthetic algae including Euglena.

Topics: Algal Proteins; Amino Acid Sequence; Animals; Ascorbic Acid; Carboxylic Ester Hydrolases; Escherichia coli; Euglena gracilis; Hexuronic Acids; Molecular Sequence Data; Protozoan Proteins; Rats; Recombinant Proteins; Sugar Acids

D-Galacturonic acid reductase, a key enzyme in ascorbate biosynthesis, was purified to homogeneity from Euglena gracilis. The enzyme was a monomer with a molecular mass of 38-39 kDa, as judged by SDS-PAGE and gel filtration. Apparently it utilized NADPH with a Km value of 62.5+/-4.5 microM and uronic acids, such as D-galacturonic acid (Km=3.79+/-0.5 mM) and D-glucuronic acid (Km=4.67+/-0.6 mM). It failed to catalyze the reverse reaction with L-galactonic acid and NADP(+). The optimal pH for the reduction of D-galacturonic acid was 7.2. The enzyme was activated 45.6% by 0.1 mM H(2)O(2), suggesting that enzyme activity is regulated by cellular redox status. No feedback regulation of the enzyme activity by L-galactono-1,4-lactone or ascorbate was observed. N-terminal amino acid sequence analysis revealed that the enzyme is closely related to the malate dehydrogenase families.

Topics: Alcohol Oxidoreductases; Amino Acid Sequence; Animals; Ascorbic Acid; Catalysis; Euglena gracilis; Hydrogen-Ion Concentration; Kinetics; Molecular Sequence Data; NAD (+) and NADP (+) Dependent Alcohol Oxidoreductases; Sequence Alignment; Substrate Specificity; Sugar Acids

The alkaloid lycorine, which is considered to inhibit the last step in ascorbic acid biosynthesis, is produced by Narcissus pseudonarcissus. The growth of two strains (C1 and C3) of Cryptococcus laurentii isolated from root tips of N. pseudonarcissus is inhibited by lycorine, as is the in vivo production of ascorbic acid from L-galactonic acid-gamma-lactone. In contrast, C. laurentii strain C4, isolated from the lycorine-containing bracts of the bulb, was not inhibited by lycorine and did not contain ascorbic acid when cultivated with or without L-galactonic acid-gamma-lactone.

Topics: Amaryllidaceae Alkaloids; Ascorbic Acid; Colony Count, Microbial; Cryptococcus; Culture Media; Gene Expression Regulation, Fungal; Lactones; Narcissus; Phenanthridines; Plant Roots; Plant Structures; Sugar Acids