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

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Abstract
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.

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