Functional characterization of D-galacturonic acid reductase, a key enzyme of the ascorbate biosynthesis pathway, from Euglena gracilis.
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.
Ishikawa
Takahiro
T
Department of Life and Environmental Sciences, Shimane University, Nishikawatsu, Matsue, Shimane, Japan. ishikawa@life.shimane-u.ac.jp
Masumoto
Ikuko
I
Iwasa
Naofumi
N
Nishikawa
Hitoshi
H
Sawa
Yoshihiro
Y
Shibata
Hitoshi
H
Nakamura
Ayana
A
Yabuta
Yukinori
Y
Shigeoka
Shigeru
S
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
D013400:Sugar Acids; C012991:galactonic acid; D000429:Alcohol Oxidoreductases; D064430:NAD (+) and NADP (+) Dependent Alcohol Oxidoreductases; C477073:D-galacturonate reductase; D001205:Ascorbic Acid