Feedback inhibition of spinach L-galactose dehydrogenase by L-ascorbate
Feedback inhibition of spinach L-galactose dehydrogenase by L-ascorbate
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DOI：
10.1093/pcp/pch152
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发表时间：
2004-09-01
影响因子：
4.9
通讯作者：
Shigeoka, S
中科院分区：
文献类型：
--
作者：
Mieda, T;Yabuta, Y;Shigeoka, S
本文研究了l -半乳糖脱氢酶（L-GalDH）的酶学性质，该酶是植物中l -抗坏血酸（AsA）生物合成途径中的关键酶。L-GalDH从菠菜叶中纯化约560倍。该酶为同二聚体，亚基质量为36 kDa。我们还克隆了菠菜L-GalDH的全长cDNA，该cDNA包含一个开放阅读框，编码322个氨基酸残基，计算分子质量为35261 Da。与猕猴桃、苹果和拟南芥的L-GalDH同源性分别为82%、79%和75%。由cDNA在大肠杆菌中表达的重组酶具有L-GalDH活性。Southern blot分析表明，菠菜L-GalDH基因出现在一个拷贝中。Northern blot分析表明，L-GalDH在菠菜的不同器官中均有表达。纯化的天然L-GalDH对l -半乳糖具有很高的特异性，K-m为116.2+/-3.2 muM。有趣的是，菠菜L-GalDH被AsA（生物合成途径的最终产物）可逆抑制。抑制动力学显示为线性竞争抑制，K-i为133.2+/-7.2 muM，表明植物对AsA合成有反馈调节。
We have studied the enzymological properties of L-galactose dehydrogenase (L-GalDH), a key enzyme in the biosynthetic pathway of L-ascorbate (AsA) in plants. L-GalDH was purified approximately 560-fold from spinach leaves. The enzyme was a homodimer with a subunit mass of 36 kDa. We also cloned the full-length cDNA of spinach L-GalDH, which contained an open reading frame encoding 322 amino acid residues with a calculated molecular mass of 35,261 Da. The deduced amino acid sequence of the cDNA showed 82, 79 and 75% homology to L-GalDH from kiwifruit, apple and Arabidopsis, respectively. Recombinant enzyme expressed from the cDNA in Escherichia coli showed L-GalDH activity. Southern blot analysis revealed that the spinach L-GalDH gene occurs in a single copy. Northern blot analysis suggests that L-GalDH is expressed in different organs of spinach. The purified native L-GalDH showed high specificity for L-galactose with a K-m of 116.2+/-3.2 muM. Interestingly, spinach L-GalDH exhibited reversible inhibition by AsA, the end-product of the biosynthetic pathway. The inhibition kinetics indicated a linear-competitive inhibition with a K-i of 133.2+/-7.2 muM, suggesting feedback regulation in AsA synthesis in the plant.