title: Feedback inhibition of spinach L-galactose dehydrogenase by L-ascorbate.
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 microM. 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 microM, suggesting feedback regulation in AsA synthesis in the plant.
authors.affiliation: Department of Food and Nutrition, Faculty of Agriculture, Kinki University, 3327-204 Nakamachi, Nara, 631-8505 Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Mieda
authors.forename: Yukinori
authors.initials: Y
authors.lastname: Yabuta
authors.forename: Madhusudhan
authors.initials: M
authors.lastname: Rapolu
authors.forename: Takashi
authors.initials: T
authors.lastname: Motoki
authors.forename: Toru
authors.initials: T
authors.lastname: Takeda
authors.forename: Kazuya
authors.initials: K
authors.lastname: Yoshimura
authors.forename: Takahiro
authors.initials: T
authors.lastname: Ishikawa
authors.forename: Shigeru
authors.initials: S
authors.lastname: Shigeoka
mesh_terms: D000595:Amino Acid Sequence; D001205:Ascorbic Acid / Q000494:pharmacology*; D001483:Base Sequence; D015139:Blotting, Southern; D017931:DNA Primers; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005691:Galactose Dehydrogenases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D018515:Plant Leaves / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D018724:Spinacia oleracea / Q000201:enzymology*
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D005691:Galactose Dehydrogenases; C047959:galactose dehydrogenase; D001205:Ascorbic Acid