Feedback inhibition of spinach L-galactose dehydrogenase by L-ascorbate

We have studied the enzymological properties of Lgalactose dehydrogenase (L-GalDH), a key enzyme in the biosynthetic pathway of L-ascorbate (AsA) in plants, lGalDH 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 sub(m) of 116.2 +- 3.2 micro M. 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 sub(I) of 133.2 +-7.2 micro M, suggesting feedback regulation in AsA synthesis in the plant.