Biochemistry and Molecular Biology of Ascorbic Acid Biosynthesis

  • Morimitsu Nishikimi
  • Kunio Yagi
Part of the Subcellular Biochemistry book series (SCBI, volume 25)


Ascorbic acid is synthesized by a variety of organisms of the animal and plant kingdoms. Among mammals, however, humans, other primates, and guinea pigs cannot exceptionally produce this vitamin, and as a consequence, they are subject to a vitamin C—deficiency disease, scurvy, if the supply of vitamin C from their diet is not sufficient. The genetic defect causing the inability to synthesize ascorbic acid in these animals arose as a result of a mutation that had occurred during their evolution, and this trait is currently carried in all individuals of the scurvy-prone species. In this sense, scurvy is an unusual type of inborn error of metabolism (Nishikimi and Udenfriend, 1977; Stone, 1967). Besides the above-mentioned scurvy-prone animals, there is a mutant rat strain that suffers from scurvy when fed a vitamin C—deficient diet (Mizushima et al., 1984). In this chapter we will focus on the genetic basis of the incapability of humans, guinea pigs, and the scurvy-prone mutant rat to biosynthesize ascorbic acid. In fact, elucidation of the human genetic defect at the gene level has long been a subject of interest for ascorbic acid research. We will also deal with the recent studies related to biosynthesis of ascorbic acid, including the terminal enzymes of the biosynthetic pathways of ascorbic acid.


Ascorbic Acid Berberine Bridge Enzyme Ascorbic Acid Synthesis Ascorbic Acid Biosynthesis Lactone Oxidase 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



base pairs


flavine adenine dinucleotide


L-gulono-γ-lactone oxidase

ODS rat

osteogenic disorder Shionogi rat


uridine diphosphate glucuronosyl-transferase


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. August, P. R., Flickinger, M. C, and Sherman, D. H., 1994, Cloning and analysis of a locus (mer) involved in mytomycin C resistance in Streptomyces lavendulae, J. Bacteriol. 176:4448–4454.PubMedGoogle Scholar
  2. Berget, S. M., 1984, Are U4 small nuclear ribonucleoproteins involved in polyadenylation?, Nature 309:179–182.PubMedCrossRefGoogle Scholar
  3. Birney, E. C, Jenness, R., and Hume, I. D., 1979, Ascorbic acid biosynthesis in mammalian kidney, Experientia 35:1425–1426.PubMedCrossRefGoogle Scholar
  4. Bleeg, H. S., and Christensen, F., 1982, Biosynthesis of ascorbate in yeast. Purification of L-galactono-1,4-lactone oxidase with properties different from mammalian L-gulonolactone oxidase, Eur. J. Biochem. 127:391–396.PubMedCrossRefGoogle Scholar
  5. Brandsch, R., 1994, A new family of flavoenzymes?, in Flavins and Flavoproteins 1993 (K. Yagi, ed.), pp. 807–810, Walter de Gruyter, Berlin.Google Scholar
  6. Bucher, P., 1990, Weight matrix descriptions of four eukaryotic RNA polymerase II promoter elements derived from 502 unrelated promoter sequences, J. Mol. Biol. 212:563–578.PubMedCrossRefGoogle Scholar
  7. Burns, J. J., 1957, Missing step in man, monkey and guinea pig required for the biosynthesis of L-ascorbic acid, Nature 180:553.PubMedCrossRefGoogle Scholar
  8. Burns, J. J., 1960, Ascorbic acid, in Metabolic Pathways, vol. 1 (D. M. Greenberg, ed.), pp. 341–356, Academic Press, New York.Google Scholar
  9. Chatterjee, I. B., 1973a, Evolution and the biosynthesis of ascorbic acid, Science 182:1271–1272.CrossRefGoogle Scholar
  10. Chatterjee, I. B., 1973b, Vitamin C synthesis in animals: Evolutionary trend, Sci. Cult. 39:210–212.Google Scholar
  11. Chatterjee, I. B., Kar, N. C, Ghosh, N. C, and Guha, B. C, 1961, Biosynthesis of L-ascorbic acid: Missing steps in animals incapable of synthesizing the vitamin, Nature 192:163–164.PubMedCrossRefGoogle Scholar
  12. Chaudhuri, C. R., and Chatterjee, I. B., 1969, L-Ascorbic acid synthesis in birds: Phylogenetic trend, Science 164:435–436.PubMedCrossRefGoogle Scholar
  13. Dabrowski, K., 1994, Primitive Actinopterigian fishes can synthesize ascorbic acid, Experientia 50:745–748.CrossRefGoogle Scholar
  14. Dykhuizen, D. E., Harrison, K. M., and Richardson, B. J., 1980, Evolutionary implications of ascorbic acid production in the Australian lungfish, Experientia 36:945–946.PubMedCrossRefGoogle Scholar
  15. Fujitsuka, N., Yokozawa, T., Oura, H., Akao, T., Kobashi, K., Ienaga, K., and Nakamura, K., 1993, L-Gulono-γ-lactone oxidase is the enzyme responsible for the production of methylguanidine in the rat liver, Nephron 63:445–451.PubMedCrossRefGoogle Scholar
  16. Grange, T., Roux, J., Rigaud, G., and Pictet, R., 1991, Cell-type specific activity of two glucocorticoid responsive units of rat tyrosine aminotransferase gene is associated with multiple binding sites for C/EBP and a novel liver-specific nuclear factor, Nucleic Acids Res. 19:131–139.PubMedCrossRefGoogle Scholar
  17. Harada, Y., Shimizu, M., Murakawa, S., and Takahashi, T., 1979, Identification of FAD of D-gluconolactone dehydrogenase: D-Erythrobic acid producing enzyme of Penicillium cyaneo-fulvum, Agric. Biol. Chem. 43:2635–3636.CrossRefGoogle Scholar
  18. Hayashida, H., and Miyata, T., 1983, Unusual evolutionary conservation and frequent DNA segment exchange in class I genes of the major histocompatibility complex, Proc. Natl. Acad. Sci. USA 80:2671–2675.PubMedCrossRefGoogle Scholar
  19. Heick, H. M. C., Graff, G. L. A., and Humpers, J. E. C., 1972, The occurrence of ascorbic acid among the yeasts, Can. J. Microbiol. 18:597–600.PubMedCrossRefGoogle Scholar
  20. Hollmann, S., and Touster, O., 1962, Alterations in tissue levels of uridine diphosphate glucose dehydrogenase, uridine diphosphate glucuronic acid pyrophosphatase and glucuronyl transferase induced by substances influencing the production of ascorbic acid, Biochim. Biophys. Acta 26:338–352.CrossRefGoogle Scholar
  21. Horio, F., and Yoshida, A., 1993, Regulatory mechanism of ascorbic acid biosynthesis stimulated by xenobiotics, Vitamins (Japan) 67:657–665.Google Scholar
  22. Horio, F., Kimura, M., and Yoshida, A., 1983, Effect of several xenobiotics on the activities of enzymes affecting ascorbic acid synthesis in rats, J. Nutr. Sci. Vitaminol. 29:233–247.PubMedGoogle Scholar
  23. Horio, F., Shibata, T., Makino, S., Machino, S., Hayashi, Y., Hattori, T., and Yoshida, A., 1993a, UDP glucuronosyltransferase gene expression is involved in the stimulation of ascorbic acid biosynthesis by xenobiotics in rats, J. Nutr. 123:2075–2084.Google Scholar
  24. Horio, F., Shibata, T., Naito, Y., Nishikimi, M., Yagi, K., and Yoshida, A., 1993b, L-Gulono-γ-lactone oxidase is not induced in rats by xenobiotics stimulating L-ascorbic acid biosynthesis, J. Nutr. Sci. Vitaminol. 39:1–9.Google Scholar
  25. Hosokawa, S., Tagaya, O., Mikami, T., Nozaki, Y., Kawaguchi, A., Yamatsu, K., and Shamoto, M., 1992, A new rat mutant with chronic conjugated hyperbilirubinemia and renal glomerular lesions, Lab. Anim. Sci. 42:27–34.PubMedGoogle Scholar
  26. Imai, T., Hirai, M., and Oba, K., 1995, Flavin is a prosthetic group of L-galactonolactone dehydrogenase from sweet potato, Plant Cell Physiol. (Suppl.), 36:140.Google Scholar
  27. Isherwood, F. A., Chen, Y. T., and Mapson, L. W., 1953, Synthesis of L-ascorbic acid in plants and animals, Nature 171:348–349.PubMedCrossRefGoogle Scholar
  28. Iyanagi, T., Watanabe, T., and Uchiyama, Y., 1989, The 3-methylcholanthrene-inducible UDP-glucuronosyltransferase deficiency in the hyperbilirubinemic rat (Gunn rat) is caused by a —1 frameshift mutation, J. Biol. Chem. 264:21302–21307.PubMedGoogle Scholar
  29. Kawai, T., Nishikimi, M., Ozawa, T., and Yagi, K., 1992, A missense mutation of L-gulono-γ-lactone oxidase causes the inability of scurvy-prone osteogenic disorder rats to synthesize L-ascorbic acid, J. Biol. Chem. 267:21973–21976.PubMedGoogle Scholar
  30. Kenney, W. C, Edmondson, D. E., Singer, T. P., Nakagawa, H., Asano, A., and Sato, R., 1976, Identification of the covalently bound flavin of L-gulono-γ-lactone oxidase, Biochem. Biophys. Res. Commun. 71:1194–1200.PubMedCrossRefGoogle Scholar
  31. Kenney, W. C, Edmondson, D. E., Singer, T. P., Nishikimi, M., Noguchi, E., and Yagi, K., 1979, Identification of the covalently-bound flavin of L-galactonolactone oxidase from yeast, FEBS Lett. 97:40–42.PubMedCrossRefGoogle Scholar
  32. Kimura, M., 1983, The Neutral Theory of Molecular Evolution, Cambridge University Press, Cambridge.Google Scholar
  33. Kiuchi, K., Nishikimi, M., and Yagi, K., 1982, Purification and characterization of L-gulonolactone oxidase from chicken kidney microsomes, Biochemistry 21:5076–5082.PubMedCrossRefGoogle Scholar
  34. Koshizaka, T., Nishikimi, M., Tanaka, M., Nakashima, K., Ozawa, T., and Yagi, K., 1987, In vitro synthesis of L-gulono-γ-lactone oxidase by rabbit reticulocyte lysate, Biochem. Int. 15:779–783.PubMedGoogle Scholar
  35. Koshizaka, T., Nishikimi, M., Ozawa, T., and Yagi, K., 1988, Isolation and sequence analysis of a complementary DNA encoding rat liver L-gulono-γ-lactone oxidase, a key enzyme for L-ascorbic acid biosynthesis, J. Biol. Chem. 263:1619–1621.PubMedGoogle Scholar
  36. Kozak, M., 1991, Structural features in eukaryotic mRNAs that modulate the initiation of translation, J. Biol. Chem. 266:19867–19870.PubMedGoogle Scholar
  37. Li, W.-H., and Tanimura, M., 1987, The molecular clock runs more slowly in man than apes and monkeys, Nature 326:93–96.PubMedCrossRefGoogle Scholar
  38. Loewus, F. A., Wagner, G., and Yang, J. C., 1975, Biosynthesis and metabolism of ascorbic acid in plants, Ann. N.Y. Acad. Sci. 258:7–23.PubMedCrossRefGoogle Scholar
  39. Loewus, M. W., Bedgar, D. L., Saito, K., and Loewus, F. A., 1990, Conversion of L-sorbosone to L-ascorbic acid by an NADP-dependent dehydrogenase in bean and spinach leaf, Plant Physiol. 94:1492–1495.PubMedCrossRefGoogle Scholar
  40. Mapson, L. W., and Breslow, E., 1958, Biological synthesis of L-ascorbic acid: L-Galactono-γ-lactone dehydrogenase, Biochem. J. 68:395–406.PubMedGoogle Scholar
  41. McLauchlan, J., Gaffney, D., Whitton, J. L., and Clements, J. B., 1985, The consensus sequence YGTGTTYY located downstream from the AATAAA signal is required for efficient formation of mRNA 3′ termini, Nucleic Acids Res. 13:1347–1368.PubMedCrossRefGoogle Scholar
  42. Miyata, T., and Yasunaga, T., 1980, Molecular evolution of mRNA: A method for estimating evolutionary rates of synonymous and amino acid substitutions from homologous nucleotide sequences and its application, J. Mol. Evol. 16:23–36.PubMedCrossRefGoogle Scholar
  43. Mizushima, Y, Harauchi, T., Yoshizaki, T., and Makino, S., 1984, A rat mutant unable to synthesize vitamin C, Experientia 40:359–361.PubMedCrossRefGoogle Scholar
  44. Nakagawa, H., and Asano, A., 1970, Ascorbate-synthesizing system in rat liver microsomes I. Gulonolactone-reducible pigment as a prosthetic group of gulonolactone oxidase, J. Biochem. (Tokyo) 68:737–746.Google Scholar
  45. Nakagawa, H., Asano, A., and Sato, R., 1975, Ascorbate-synthesizing system in rat liver microsomes II. A peptide-bound flavin as the prosthetic group of L-gulono-γ-lactone oxidase, J. Biochem. (Tokyo) 77:221–232.Google Scholar
  46. Nakajima, Y., Shantha, T. R., and Bourne, G. H., 1969, Histochemical detection of L-gulonolactone: Phenazine methosulfate oxidoreductase activity in several mammals with special reference to synthesis of vitamin C in primates, Histochemie 18:293–301.PubMedGoogle Scholar
  47. Nakamura, T., Satoh, T., Horie, T., Sagami, F., and Tagaya, O., 1989, Strain differences of rat liver carboxylesterase activities related to the phenotype difference of esterase-3 (egasyn), Res. Commun. Chem. Pathol. Pharmacol. 66:451–459.PubMedGoogle Scholar
  48. Nei, M., and Gojobori, T., 1986, Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions, Mol. Biol. Evol. 3:418–426.PubMedGoogle Scholar
  49. Nishikimi, M., and Udenfriend, S., 1976, Immunologic evidence that the gene for L-gulono-γ-lactone oxidase is not expressed in animals subject to scurvy, Proc. Natl. Acad. Sci. USA 73:2066–2068.PubMedCrossRefGoogle Scholar
  50. Nishikimi, M., and Udenfriend, S., 1977, Scurvy as an inborn error of ascorbic acid biosynthesis, Trends Biochem. Sci. 2:111–113.CrossRefGoogle Scholar
  51. Nishikimi, M., Tolbert, B. M., and Udenfriend, S., 1976., Purification and characterization of L-gulono-γ-lactone oxidase from rat and goat liver, Arch. Biochem. Biophys. 175:427–435.PubMedCrossRefGoogle Scholar
  52. Nishikimi, M., Noguchi, E., and Yagi, K., 1978, Occurrence in yeastof L-galactonolactone oxidase which is similar to a key enzyme for ascorbic acid biosynthesis in animals, L-gulonolactone oxidase, Arch. Biochem. Biophys. 191:479–486.PubMedCrossRefGoogle Scholar
  53. Nishikimi, M., Koshizaka, T., Mochizuki, H., Iwata, H., Makino, S., Hayashi, Y., Ozawa, T., and Yagi, K., 1988a, L-Gulono-γ-lactone oxidase deficiency in rats with osteogenic disorder: Enzymological and immunochemical studies, Biochem. Int. 16:615–621.Google Scholar
  54. Nishikimi, M., Koshizaka, T., Ozawa, T., and Yagi, K., 1988b, Occurrence in humans and guinea pigs of the gene related to their missing enzyme L-gulono-γ-lactone oxidase, Arch. Biochem. Biophys. 267:842–846.CrossRefGoogle Scholar
  55. Nishikimi, M., Koshizaka, T., Kondo, K., Ozawa, T., and Yagi, K., 1989, Expression of the mutant gene for L-gulono-γ-lactone oxidase in scurvy–prone rats, Experientia 45:126–129.PubMedCrossRefGoogle Scholar
  56. Nishikimi, M., Kawai, T., Ozawa, T., and Yagi, K., 1992, Guinea pigs possess a highly mutated gene for L-gulono-γ-lactone oxidase, the key enzyme for L-ascorbic acid biosynthesis missing in this species, J. Biol. Chem. 267:21967–21972.PubMedGoogle Scholar
  57. Nishikimi, M., Fukuyama, R., Minoshima, S., Shimizu, N., and Yagi, K., 1994a, Cloning and chromosomal mapping of the human nonfunctional gene for L-gulono-γ-lactone oxidase, the enzyme for L-ascorbic acid biosynthesis missing in man, J. Biol. Chem. 269:13685–13688.Google Scholar
  58. Nishikimi, M., Kobayashi, J., and Yagi, K., 1994b, Production by a baculovirus expression system of the apoprotein of L-gulono-γ-lactone oxidase, a flavoenzyme possessing a covalently bound FAD, Biochem. Mol. Biol. Int. 33:313–320.Google Scholar
  59. Oba, K., Fukui, M., Imai, Y., Iriyama, S., and Nogami, K., 1994, L-Galactono-γ-lactonedehydrogenase: Partial characterization, induction of activity and role in the synthesis of ascorbic acid in wounded white potato tuber tissue, Plant Cell Physiol. 35:473–478.Google Scholar
  60. Oba, K., Ishikawa, S., Nishikawa, M., Mizuno, H., and Yamamoto, T., 1995, Purification and properties of L-galactono-γ-lactone dehydrogenase, a key enzyme for ascorbic acid biosynthesis, from sweet potato roots, J. Biochem. (Tokyo) 117:120–124.Google Scholar
  61. Ramji, D. P., Tadros, M. H., Hardon, E. M., and Cortese, R., 1991, The transcription factor LF-A1 interacts with a bipartite recognition sequence in the promoter regions of several liver-specific genes, Nucleic Acids Res. 19:1139–1146.PubMedCrossRefGoogle Scholar
  62. Saito, K., Nick, J. A., and Loewus, F. A., 1990, D-Glucosone and L-sorbosone, putative intermediates of L-ascorbic acid biosynthesis in detached bean and spinach leaves, Plant Physiol. 94:1496–1500.PubMedCrossRefGoogle Scholar
  63. Sato, P., and Grahn, I., 1981, Administration of chicken L-gulonolactone oxidase to guinea pigs evokes ascorbic acid synthetic capacity, Arch. Biochem. Biophys. 210:609–616.PubMedCrossRefGoogle Scholar
  64. Sato, P., and Udenfriend, S., 1978, Scurvy-prone animals, including man, monkey, and guinea pig do not express the gene for gulonolactone oxidase, Arch. Biochem. Biophys. 187:158–162.PubMedCrossRefGoogle Scholar
  65. Sato, P., Nishikimi, M., and Udenfriend, S., 1976, Is L-gulonolactone oxidase the only enzyme missing in animals subject to scurvy?, Biochem. Biophys. Res. Commun. 71:293–299.PubMedCrossRefGoogle Scholar
  66. Shigeoka, S., Nakano, Y., and Kitaoka, S., 1979, The biosynthetic pathway of L-ascorbic acid in Euglena gracilis Z, J. Nutr. Sci. Vitaminol. 25:299–307.PubMedGoogle Scholar
  67. Shimazono, N., and Mano, Y., 1961, Enzymatic studies on the metabolism of uronic and aldonic acids related to L-ascorbic acid in animal tissues, Ann. N.Y. Acad. Sci. 92:91–104.CrossRefGoogle Scholar
  68. Stone, I., 1967, The genetic disease, hypoascorbemia. A fresh approach to an ancient disease and some of its medical implications, Acta Genet. Med. Gemellol. 16:52–62.PubMedGoogle Scholar
  69. Swimmer, C, and Shenk, T, 1985, Selection of sequence elements that substitute for the standard AATAAA motif which signals 3′ processing and polyadenylation of late simian virus 40 mRNAs, Nucleic Acids Res. 13:8053–8063.PubMedCrossRefGoogle Scholar
  70. Takahashi, T., Yamashita, H., Kato, E., Mitsumoto, M., and Murakawa, S., 1976, Purification and some properties of D-glucono-γ-lactone dehydrogenase. D-Erythrobic acid producing enzyme of Penicillium cyaneo–fulvum, Agric. Biol. Chem. 40:121–129.CrossRefGoogle Scholar
  71. Thomas, P., Bally, M. B., and Neff, J. M., 1985, Influence of some environmental variables on the ascorbic acid status of mullet, Mugil cephalus L., tissues II. Seasonal fluctuations and biosynthetic ability, J. Fish Biol. 27:47–57.CrossRefGoogle Scholar
  72. Touhata, K., Toyohara, H., Kinoshita, M., Mitani, T, Sato, M., and Sakaguchi, M., 1993, Molecular evolution of gulonolactone oxidase in vertebrates II. Purification of the enzyme from lamprey kidney, in Abstracts for the Meetings of Japanese Society of Scientific Fisheries (October), p. 196.Google Scholar
  73. Toyohara, H., Touhata, K., Kinoshita, M., Mitani, T., Sato, M., Sakaguchi, M., Nishikimi, M., and Yagi, K., 1992, Molecular evolution of L-gulonolactone oxidase in vertebrates I. Its distribution among vertebrates, in Abstracts for the Meetings of Japanese Society of Scientific Fisheries (October), p. 150.Google Scholar
  74. Wilson, R. P., 1973, Absence of ascorbic acid synthesis in channel catfish, Ictaluruspunctatus and blue catfish, Ictalurus jmeatus, Comp. Biochem. Physiol. 46B:635–638.CrossRefGoogle Scholar
  75. Winkelman, J., and Lehninger, A. L., 1958, Aldono– and uronolactonase of animal tissues, J. Biol. Chem. 233:794–799.PubMedGoogle Scholar
  76. Yagi, K., Koshizaka, T., Kito, M., Ozawa, T., and Nishikimi, M., 1991, Expression in monkey cells of the missing enzyme in L-ascorbic acid biosynthesis, L-gulono-γ-lactone oxidase, Biochem. Biophys. Res. Commun. 177:659–663.PubMedCrossRefGoogle Scholar
  77. Yamamoto, Y., Sato, M., and Ikeda, S., 1978, Existence of L-gulonolactone oxidase in some teleosts, Bull. Jpn. Soc. Sci. Fish. 44:775–779.Google Scholar

Copyright information

© Plenum Press, New York 1996

Authors and Affiliations

  • Morimitsu Nishikimi
    • 1
  • Kunio Yagi
    • 1
  1. 1.Institute of Applied BiochemistryGifu, MitakeJapan

Personalised recommendations