Ishikawa Takahiro, Masumoto Ikuko, Iwasa Naofumi, Nishikawa Hitoshi, Sawa Yoshihiro, Shibata Hitoshi, Nakamura Ayana, Yabuta Yukinori, Shigeoka Shigeru
Department of Life and Environmental Sciences, Shimane University, Nishikawatsu, Matsue, Shimane, Japan.
Biosci Biotechnol Biochem. 2006 Nov;70(11):2720-6. doi: 10.1271/bbb.60327. Epub 2006 Nov 7.
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.
D-半乳糖醛酸还原酶是抗坏血酸生物合成中的关键酶，已从纤细裸藻中纯化至同质。通过SDS-PAGE和凝胶过滤判断，该酶为单体，分子量为38 - 39 kDa。显然，它利用NADPH，其Km值为62.5±4.5 μM，以及糖醛酸，如D-半乳糖醛酸（Km = 3.79±0.5 mM）和D-葡萄糖醛酸（Km = 4.67±0.6 mM）。它不能催化L-半乳糖酸与NADP(+)的逆反应。还原D-半乳糖醛酸的最适pH为7.2。该酶被0.1 mM H(2)O(2)激活45.6%，表明酶活性受细胞氧化还原状态调节。未观察到L-半乳糖-1,4-内酯或抗坏血酸对酶活性的反馈调节。N端氨基酸序列分析表明，该酶与苹果酸脱氢酶家族密切相关。
