Purification and properties of L-galactono-gamma-lactone dehydrogenase, a key enzyme for ascorbic acid biosynthesis, from sweet potato roots

• Published in: Journal of biochemistry (Tokyo) Vol. 117; no. 1; pp. 120 - 124
• Main Authors: Oba, K, Ishikawa, S, Nishikawa, M, Mizuno, H, Yamamoto, T
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 1995
• Subjects: ascorbic acid; Ascorbic Acid - biosynthesis; biosynthesis; enzyme activity; Enzyme Stability; galactose; Ipomoea batatas; Kinetics; L-ascorbic acid; L-galactono-γ-lactone dehydrogenase; L-gulono-γ-lactone oxidase; lactones; Mitochondria - enzymology; Molecular Weight; oxidoreductases; Oxidoreductases - chemistry; Oxidoreductases - isolation & purification; Oxidoreductases Acting on CH-CH Group Donors; Plant Roots - enzymology; plants; purification; Substrate Specificity; substrates; sweet potato (Ipomoea batatas); sweet potatoes; Vegetables - enzymology
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/oxfordjournals.jbchem.a124697

L-Galactono-gamma-lactone dehydrogenase (L-galactono-gamma-lactone:ferricytochrome c oxidoreductase [EC 1.3.2.3], GLDHase) which catalyzes the terminal step in the biosynthesis of L-ascorbic acid (ABA) has been purified from roots of sweet potato (Ipomaea batatas L., cv. Kintoki). Highly purified preparation of the GLDHase was obtained by three column chromatography steps with a recovery of ca. 1%, after solubilization from mitochondria in sweet potato roots. SDS-PAGE exhibited a single band at 56 kDa. In the native state, the apparent molecular mass of the enzyme was 56 kDa, based on a Sephadex G-100 gel filtration. The pI and optimum pH values were 5.8 and 7.9, respectively. The Km value for L-galactono-gamma-lactone was 0.12 mM. Substrate inhibition was obtained at concentrations greater than 4.2 mM. The enzyme was inhibited by p-chloromercuribenzoate (PCMB) and acriflavine, and the inhibition of acriflavine was diminished by the addition of FAD or FMN. The only effective substrate for the GLDase was L-galactono-gamma-lactone.