NADP+-dependent D-arabinose dehydrogenase shows a limited contribution to erythroascorbic acid biosynthesis and oxidative stress resistance in Saccharomyces cerevisiae
NADP+-dependent D-arabinose dehydrogenase shows a limited contribution to erythroascorbic acid biosynthesis and oxidative stress resistance in Saccharomyces cerevisiae
复制标题
DOI：
10.1271/bbb.60399
复制
发表时间：
2006-12-01
影响因子：
1.6
通讯作者：
Goda, Kiyoshi
中科院分区：
文献类型：
--
作者：
Amako, Katsumi;Fujita, Kazuyo;Goda, Kiyoshi
研究了NADP（+）依赖性d -阿拉伯糖脱氢酶（ARA）的分子特征和生理意义，该酶被认为在酵母中抗坏血酸类似物d -红抗坏血酸的生物合成中起作用。在大肠杆菌中产生的ARA的一个大亚基Ara1p被纯化为同型二聚体，其中一些在n端被降解。显示有足够的ARA活性。Ara1p的降解在酵母细胞中自然发生，ARA的小亚基之前被认为实际上是Ara1p的自然降解产物。ARA1缺陷突变体几乎失去了所有NADP(+)-ARA活性，但细胞内d -红抗坏血酸仅减少一半。该突变体对H2O2和二胺的敏感性增加，但对美萘醌和过氧化叔丁基没有敏感性。给突变细胞喂食d -阿拉伯糖导致细胞内d -红抗坏血酸增加，表明存在另一种ARA同工酶。补充d -阿拉伯糖后，ARA1突变体恢复了对H2O2的抗性。我们的研究结果表明，Ara1p对d -红抗坏血酸的生物合成和氧化应激抵抗的直接贡献是非常有限的。
The molecular aspects and physiological significance of NADP(+)-dependent D-arabinose dehydrogenase (ARA), which is thought to function in the biosynthesis of an analog of ascorbic acid, D-erythroascorbic acid in yeasts, were examined. A large subunit of ARA, Ara1p produced in E. coli, was purified as a homodimer, some of which was degraded at the N-terminus. It showed sufficient ARA activity. Degradation of Ara1p occurs naturally in yeast cells, and the small subunit of ARA previously thought as is, in fact, a naturally occuring degradation product of Ara1p. A deficient mutant of ARA1 lost almost all NADP(+)-ARA activity, but intracellular D-erythroascorbic acid was only halved. This mutant showed increased susceptibility to H2O2 and diamide but not to menadione or tert-butylhydroperoxide. Feeding D-arabinose to mutant cells led to increases in intracellular D-erythroascorbic acid, suggesting the presence of another ARA isozyme. The deficient mutant of ARA1 recovered resistance to H2O2 with feeding Of D-arabinose. Our results suggest that the direct contributions of Ara1p both to D-erythroascorbic acid biosynthesis and to oxidative stress resistance are quite limited.