Molecular characterization of a novel glucose-6-phosphate dehydrogenase from potato (Solanum tuberosum L.).
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 Km values determined for both substrates were significantly lower compared to those of P1. The high Vmax and Ki [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.
Wendt
U K
UK
Pflanzenphysiologie, FB5 Biologie/Chemie, Universität Osnabrück, Barbarastrasse 11, 49076 Osnabrück, Germany.
Wenderoth
I
I
Tegeler
A
A
Von Schaewen
A
A
D000595:Amino Acid Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D018076:DNA, Complementary; D005954:Glucosephosphate Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D011198:Solanum tuberosum / Q000201:enzymology / Q000235:genetics*
D018076:DNA, Complementary; D011994:Recombinant Proteins; D005954:Glucosephosphate Dehydrogenase