Functional Characterization of D-Galacturonic Acid Reductase, a Key Enzyme of the Ascorbate Biosynthesis Pathway, from Euglena gracilis

• Published in: Bioscience, biotechnology, and biochemistry biotechnology, and biochemistry, 2006-11, Vol.70 (11), p.2720-2726
• Main Authors: ISHIKAWA, Takahiro, MASUMOTO, Ikuko, IWASA, Naofumi, NISHIKAWA, Hitoshi, SAWA, Yoshihiro, SHIBATA, Hitoshi, NAKAMURA, Ayana, YABUTA, Yukinori, SHIGEOKA, Shigeru
• Format: Article
• Language: English
• Subjects: Alcohol Oxidoreductases - chemistry; Alcohol Oxidoreductases - isolation & purification; Alcohol Oxidoreductases - metabolism; aldo-keto reductase; Amino Acid Sequence; Animals; ascorbate biosynthesis; Ascorbic Acid - biosynthesis; Biological and medical sciences; Catalysis; D-galacturonic acid reductase; Euglena; Euglena gracilis; Euglena gracilis - enzymology; Fundamental and applied biological sciences. Psychology; Hydrogen-Ion Concentration; Kinetics; Molecular Sequence Data; NAD (+) and NADP (+) Dependent Alcohol Oxidoreductases; Sequence Alignment; Substrate Specificity; Sugar Acids - metabolism
• ISSN: 0916-8451; 1347-6947
• DOI: 10.1271/bbb.60327

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 μM 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.