Molecular characterization of a novel glucose-6-phosphate dehydrogenase from potato (Solanum tuberosum L.)

Autor(en): Wendt, UK
Wenderoth, I
Tegeler, A
von Schaewen, A
Stichwörter: ASSIMILATION; CHLOROPLASTS; DITHIOTHREITOL; expression; glucose-6-phosphate dehydrogenase; IN-VIVO; isoenzymes; kinetic parameters; METABOLISM; Plant Sciences; PLANT-TISSUES; plastidic; PLASTIDIC GLUCOSE-6-PHOSPHATE-DEHYDROGENASE; PURIFICATION; redox regulation; ROOT PLASTIDS; STREPTAVIDIN
Erscheinungsdatum: 2000
Herausgeber: WILEY
Volumen: 23
Ausgabe: 6
Startseite: 723
Seitenende: 733
We describe a novel G6PD cDNA from potato. The deduced amino acid sequence shares 77% identity with the known chloroplast enzyme, but only 47% with the corresponding cytosolic G6PDH. The sequence comprises the two cysteine residues conserved in other redox-regulated chloroplast G6PDH and a transit peptide capable of directing a GFP fusion protein to chloroplasts, demonstrating that the cDNA codes for a second plastidic G6PD isoform. The mature part was expressed in E. coli. When synthesized with a C-terminal Strep tag, the enzyme retained G6PDH activity upon affinity purification. In the presence of reductively activated spinach thioredoxin, G6PDH activity decreased by about 50%. This protein-mediated activity loss was completely reversed by addition of oxidant. In contrast to the chloroplast enzyme (P1), the presence of reduced dithiothreitol alone destroyed the activity of the new G6PDH (P2), and incubation with GSH had no effect. The K-m values determined for both substrates were significantly lower compared to those of P1. The high V-max and K-i ([NADPH]) values indicate that the P2 enzyme is more active than P1 and less susceptible to feedback inhibition by its product NADPH. At the level of mRNA accumulation, differences between the two plastid-localized isoforms are most prominent in roots and growing tissues. Immunoblot analyses of isolated plastid preparations revealed that the two plastidic enzymes are present in both root and leaf tissue. The data obtained indicate that we have characterized a second plastidic G6PDH with distinct biochemical features.
ISSN: 09607412
DOI: 10.1046/j.1365-313x.2000.00840.x

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