ascorbic-acid and gulose

Vitamin C (VC) is an essential nutrient for many animals. However, whether insects, including Bombyx mori, can synthesize VC remains unclear. In this article, the optimized HPLC method was used to determine the content of l-ascorbic acid (AsA) in silkworm eggs, larvae and pupae, and the activity of l-gulono-1,4-lactone oxidase (GULO), a key enzyme in VC synthesis. The RNA interference method was used to determine the effect of the BmGulo-like gene on embryonic development and GULO activity in the pupal fat body. The AsA content increased significantly during E144 h-E168 h in the late embryonic stage and P48 h-P144 h in the middle-late pupal stage, in which exogenous VC was not ingested. Furthermore, the body AsA content in larvae fed VC-free feed also increased with larval stage. The GULO enzymatic activity was present in eggs and the fat bodies of larvae and pupae, even when the larvae were reared with fresh mulberry leaves. Moreover, the activity was higher in the later embryonic stages (E144 h-E168 h) and the early pupal stage (before P24 h). The GULO activity in the pupal fat body dramatically decreased when the screened BmGulo-like gene (BGIBMGA005735) was knocked down with small interfering RNA; in addition, the survival rate and hatching rate of eggs significantly decreased 21% and 44%, respectively, and embryonic development was delayed. Thus, Bombyx mori can synthesize AsA through the l-gulose pathway, albeit with low activity, and this synthesis ability varies with developmental stages.

Topics: Animals; Ascorbic Acid; Bombyx; Hexoses; Larva; Pupa; Sugar Acids

L-Gulose is a very rare sugar, but appears as a sugar component of the main polar lipids characteristic in such a thermophilic archaeon as Thermoplasma acidophilum that lives without cell walls in a highly acidic environment. The biosynthesis of L-gulose in this thermophilic organism was investigated with deuterium-labeling experiments. L-Gulose was found to be biosynthesized from D-glucose via stepwise stereochemical inversion at C-2 and C-5. The involvement of an epimerase related to GDP-mannose 3,5-epimerase, the key enzyme of plant ascorbate biosynthesis, was also suggested in this C-5 inversion. The resemblance of L-gulose biosynthesis in archaea and plants might be suggested from these results.

Topics: Ascorbic Acid; Glucose; Hexoses; Lipids; Plants; Stereoisomerism; Thermoplasma

Ketogulonicigenium vulgare DSM 4025, known as a 2-keto-L-gulonic acid producing strain from L-sorbose via L-sorbosone, surprisingly produced L-ascorbic acid from D-sorbitol, L-sorbose, L-gulose, and L-sorbosone as the substrate under a growing or resting condition. As the best result, K. vulgare DSM 4025 produced 1.37 g per liter of L-AA from 5.00 g per liter of L-sorbosone during 4 h incubation time at 30 degrees C under the resting cell condition having 5.70 g per liter of wet cells. The precursor of L-AA formation from D-sorbitol and L-sorbose, except for L-gulose, was thought to be the putative furanose form of L-sorbosone. This is the first time it is reported that bacteria can produce vitamin C via L-sorbosone.

Topics: Ascorbic Acid; Gluconobacter oxydans; Hexoses; Sorbitol; Sorbose

Despite its importance for agriculture, bioindustry, and nutrition, the fundamental process of L-ascorbic acid (vitamin C) biosynthesis in plants is not completely elucidated, and little is known about its regulation. The recently identified GDP-Man 3',5'-epimerase catalyzes a reversible epimerization of GDP-D-mannose that precedes the committed step in the biosynthesis of vitamin C, resulting in the hydrolysis of the highly energetic glycosyl-pyrophosphoryl linkage. Here, we characterize the native and recombinant GDP-Man 3',5'-epimerase of Arabidopsis thaliana. GDP and GDP-D-glucose are potent competitive inhibitors of the enzyme, whereas GDP-L-fucose gives a complex type of inhibition. The epimerase contains a modified version of the NAD binding motif and is inhibited by NAD(P)H and stimulated by NAD(P)+. A feedback inhibition of vitamin C biosynthesis is observed apparently at the level of GDP-Man 3',5'-epimerase. The epimerase catalyzes at least two distinct epimerization reactions and releases, besides the well known GDP-l-galactose, a novel intermediate: GDP-L-gulose. The yield of the epimerization varies and seems to depend on the molecular form of the enzyme. Both recombinant and native enzymes co-purified with a Hsp70 heat-shock protein (Escherichia coli DnaK and A. thaliana Hsc70.3, respectively). We speculate, therefore, that the Hsp70 molecular chaperones might be involved in folding and/or regulation of the epimerase. In summary, the plant epimerase undergoes a complex regulation and could control the carbon flux into the vitamin C pathway in response to the redox state of the cell, stress conditions, and GDP-sugar demand for the cell wall/glycoprotein biosynthesis. Exogenous L-gulose and L-gulono-1,4-lactone serve as direct precursors of l-ascorbic acid in plant cells. We propose an L-gulose pathway for the de novo biosynthesis of vitamin C in plants.

Topics: Amino Acid Motifs; Arabidopsis; Ascorbic Acid; Binding, Competitive; Carbohydrate Epimerases; Carbon; Chromatography, Affinity; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Glutathione Transferase; Guanosine Diphosphate Sugars; Hexoses; HSP70 Heat-Shock Proteins; Kinetics; Models, Biological; Models, Chemical; Nitrilotriacetic Acid; Oxidation-Reduction; Peptides; Plasmids; Protein Folding; Recombinant Proteins; Time Factors