title: Functional characterization of D-galacturonic acid reductase, a key enzyme of the ascorbate biosynthesis pathway, from Euglena gracilis.
abstract: 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.
authors.affiliation: Department of Life and Environmental Sciences, Shimane University, Nishikawatsu, Matsue, Shimane, Japan. ishikawa@life.shimane-u.ac.jp
authors.forename: Takahiro
authors.initials: T
authors.lastname: Ishikawa
authors.forename: Ikuko
authors.initials: I
authors.lastname: Masumoto
authors.forename: Naofumi
authors.initials: N
authors.lastname: Iwasa
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Nishikawa
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Sawa
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Shibata
authors.forename: Ayana
authors.initials: A
authors.lastname: Nakamura
authors.forename: Yukinori
authors.initials: Y
authors.lastname: Yabuta
authors.forename: Shigeru
authors.initials: S
authors.lastname: Shigeoka
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001205:Ascorbic Acid / Q000096:biosynthesis*; D002384:Catalysis; D005056:Euglena gracilis / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D064430:NAD (+) and NADP (+) Dependent Alcohol Oxidoreductases; D016415:Sequence Alignment; D013379:Substrate Specificity; D013400:Sugar Acids / Q000378:metabolism
chemical_list: D013400:Sugar Acids; C012991:galactonic acid; D000429:Alcohol Oxidoreductases; D064430:NAD (+) and NADP (+) Dependent Alcohol Oxidoreductases; C477073:D-galacturonate reductase; D001205:Ascorbic Acid