Journal Article

Feedback Inhibition of Spinach <span class="smallCaps">l</span>-Galactose Dehydrogenase by <span class="smallCaps">l</span>-Ascorbate

Takahiro Mieda, Yukinori Yabuta, Madhusudhan Rapolu, Takashi Motoki, Toru Takeda, Kazuya Yoshimura, Takahiro Ishikawa and Shigeru Shigeoka

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 45, issue 9, pages 1271-1279
Published in print September 2004 | ISSN: 0032-0781
Published online September 2004 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pch152
Feedback Inhibition of Spinach l-Galactose Dehydrogenase by l-Ascorbate

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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.

Keywords: Keywords: Ascorbate biosynthesis — Feedback inhibition — l-Galactose dehydrogenase — Spinach (Spinacia oleracea).; Abbreviations: AOS, active oxygen species; AsA, l-ascorbate; APX, ascorbate peroxidase; DTT, dithiothreitol; LB medium, Luria–Bertani medium; l-Gal, l-galactose; l-GalDH, l-galactose dehydrogenase; IPTG, isopropyl β-d-thiogalactopyranoside; SSC, standard saline citrate.

Journal Article.  5802 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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