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Plant and Cell Physiology 2004 45(9):1271-1279; doi:10.1093/pcp/pch152
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© 2004 Oxford University Press

Feedback Inhibition of Spinach L-Galactose Dehydrogenase by L-Ascorbate

Takahiro Mieda1, Yukinori Yabuta1, Madhusudhan Rapolu1, Takashi Motoki1, Toru Takeda1, Kazuya Yoshimura1, Takahiro Ishikawa2 and Shigeru Shigeoka1,3

1 Department of Food and Nutrition, Faculty of Agriculture, Kinki University, 3327-204 Nakamachi, Nara, 631-8505 Japan
2 Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue, Shimane, 690-8504 Japan

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 Km of 116.2±3.2 µM. 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 Ki of 133.2±7.2 µM, suggesting feedback regulation in AsA synthesis in the plant.

3 Corresponding author: E-mail, shigeoka{at}nara.kindai.ac.jp; Fax, +81-742-43-2252.


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